Escherichia coli strain to modify chrysazin to chrysazin-8-O-α-l-rhamnoside (CR) and chrysazin-8-O-α-l-2′-O-methylrhamnoside (CRM) using rhamnosyl transferase and sugar-O-methyltransferase. Biosynthesized CR and CRM were structurally characterized using HPLC, high-resolution mass spectrometry, and various nuclear magnetic resonance analyses. Antimicrobial effects of chrysazin, CR, and CRM against 18 superbugs, including 14 Gram-positive and 4 Gram-negative pathogens, were investigated. CR and CRM exhibited antimicrobial activities against nine pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) in a disk diffusion assay at a concentration of 40 µg per disk. There were MIC and MBC values of 7.81–31.25 µg/mL for CR and CRM against methicillin-sensitive S. aureus CCARM 0205 (MSSA) for which the parent chrysazin is more than >1000 µg/mL. Furthermore, the anti-proliferative properties of chrysazin, CR, and CRM were assayed using AGS, Huh7, HL60, and HaCaT cell lines. CR and CRM showed higher antibacterial and anticancer properties than chrysazin.Anthraquinone and its derivatives show remarkable biological properties such as anticancer, antibacterial, antifungal, and antiviral activities. Hence, anthraquinones derivatives have been of prime interest in drug development. This study developed a recombinant  Quinones are naturally occurring organic compounds found in higher plants, fungi, bacteria, and animals. They have a lot of structural varieties. Since they are found in different colors in nature, they are considered pigments . Anthraq3/P2O3Cl4 [Synthesis of anthraquinone derivatives is of great interest recently. There are various methods for the synthesis of anthraquinones derivatives, including intramolecular condensation of aryl and o-aroylbenzoic acid using fuming sulfuric acid, benzoyl chloride, concentrated sulfuric acid, benzoyl chloride, zinc chloride, and POCl/P2O3Cl4 . The che/P2O3Cl4 .Modification of anthraquinones can be performed by glycosylation, methylation, sulfation, prenylation, and so on. Glycosylation is an important process for increasing the solubility of hydrophobic compounds, improving stability, reducing toxicity, and modifying biological activities ,16. MethO-methyltransferase (OMT) that catalyzes the transfer of the methyl group of S-adenosyl-l-methionine (SAM) to hydroxyl groups [Methyltransferases are important enzymes in the modification of different substrate. However, methylation of sugar is very rare ,21. Genel groups ,22,23.Rheum palmatum L. (Polygonaceae) [Chrysazin  has been used as a medicine since ancient times. It can be found naturally. It is isolated from the root and rhizome of onaceae) . It has onaceae) ,25.E. coli sugar (thymidine diphosphate (TDP)-rhamnose) as a sugar donor, which will be used as a glycosyltransferase to conjugate to chrysazin and SAM as an O-methyltransferase to conjugate chrysazin rhamnoside. For the production of chrysazin derivatives, anthraquinone rhamnosyltransferase (7665) [Saccharothrix espanaensis and O-methyltransferase (ThnM1) [Nocardia sp. CS682 were cloned and heterologously expressed in E. coli. The recombinant strain E. coli was utilized for the production of chrysazin-8-O-α-l-rhamnoside (CR) and chrysazin-8-O-α-l-2′-O-methylrhamnoside (CRM) from the substrate chrysazin (This study is based on the utilization of indigenous e (7665)  from Sac (ThnM1)  from Nochrysazin . Anticanβ-d-1-thiogalactoside (IPTG) was purchased from GeneChem Inc. . SAM was purchased from Sigma-Aldrich . Escherichia coli BL21 (DE3)  was used as an expression and biotransformation host. Luria–Bertani (LB) broth medium and agar plates with appropriate antibiotics  were used for culture preparation, colony selection, and biotransformation. Pathogenic strains such as Staphylococcus. aureus CCARM 3640 (MRSA), S. aureus CCARM 3089 (MRSA), S. aureus CCARM 33591(MRSA), S. aureus CCARM 0205 (MSSA), S. aureus CCARM 0204 (MSSA), S. aureus CCARM 0027 (MSSA), S. aureus CCARM 3090 (MRSA), S. aureus CCARM 3634 (MRSA), S. aureus CCARM 3635 (MRSA), Bacillus subtilis ATCC 6633, Enterococcus faecalis 19433, Enterococcus faecalis 19434, Kocuria rhizophilla NBRC 12708, Micrococcus luteus, Escherichia coli ATCC 25922, Proteus hauseri NBRC 3851, Klebsiella pneumonia ATCC10031, and Salmonella enterica ATCC 14,028 were obtained from Professor Seung-Young Kim  [Chrysazin/Dantron was purchased from Tokyo Chemical Industry . HPLC-grade acetonitrile and water were purchased from Mallinckrodt Baker . All other chemicals used were of high analytical grade and commercially available. Isopropyl-E. coli, anthraquinone rhamnosyltransferase (7665) [S. espanaensis and rhamnose methyltransferase (ThnM1) [Nocardia sp. CS682 were taken to prepare E. coli S2 [E. coli S2 was generated by transforming pET32a ± 7665(Am) CRISPRi-S1(Cmr) and pCDFDuet-metK-thnM1(Sm) into an E. coli strain harboring the rhamnose cassette piBR181-tgs.dh.ep.kr.pgm2.glf.glk (Km) [For the production of rhamnosylated and methoxy-rhamnosylated derivatives of chrysazin using engineered e (7665)  from S.  (ThnM1)  from Noc coli S2 . E. coliglk (Km) . RecombiE. coli S2 was cultured in a 5 mL LB medium supplemented with ampicillin, kanamycin, chloramphenicol, and streptomycin (each at 50 μg/mL) and incubated at 37 °C for 3 h. From the 5 mL seed culture, 500 µL was transferred into a flask containing 50 mL LB broth with respective antibiotics and cultured at 37 °C for around 4 h until the optical density of cells at 600 nm (OD600) reached 0.6–0.8. This culture had added to it 0.5 mm isopropyl β-d-1-thiogalactopyranoside (IPTG) to induce protein expression, followed by incubation at 20 °C for 20 h. To determine optimal substrate concentration, glucose concentration, and time, different concentrations  of chrysazin and different concentrations  of sterile glucose solution were fed into the cell culture after induction. After 20 h of adding IPTG, chrysazin dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 400 µM was added along with 10% glucose. Samples (3 mL of culture broths) were withdrawn at 6, 12, 24, 36, 48, and 60 h for product analysis. After 48 h, compounds were extracted using a double volume of ethyl acetate  and a Soxhlet extractor. The Soxhlet extractor was kept still to separate the mixture for 4–6 h at room temperature after shaking. Ethyl acetate was removed under reduced pressure and dissolved in methanol. This sample was further analyzed by HPLC and mass spectrometry. To collect a sample to characterize structurally, the biotransformation experiment was performed using a fermenter (3 L of culture). The pure fraction of the compound was collected via preparatory-high-pressure liquid chromatography (prep-HPLC).A seed culture of 18 column  ). The binary mobile phase was composed of solvent A (HPLC-grade water + 0.1% trifluoroacetic acid) and solvent B . The total flow rate was maintained at 1 mL/min for the 30 min program. The ACN concentration began with 10%. A linear gradient from 10 to 50% for 10 min, 50–90% for 23 min, and 90–10% for 30 min was then used. The HR-QTOF ESI/MS was performed in positive ion mode using an Acquity mass spectrometer , which was coupled with a Synapt G2-S system (Waters). Purification of compounds was performed using a prep-HLPC instrument equipped with a YMC-Pack ODS-AQ C18 column,   and a connected UV detector (420 nm). Here, a 40 min binary program with implementation of 20% (0–5 min), 50% (5–10 min), 70% (10–20 min), 90% (20–25 min), 20% (25–30 min), and 10% (30–35 min) ACN at a flow rate of 10 mL/min was used. Purified products were pooled, dried, and lyophilized to remove water or moisture. Furthermore, the fully dried pure compound was dissolved in DMSO-d6 and subjected to a 700 MHz NMR spectrometer equipped with TCI CryoProbe (5 mm). From the extracted compound, a 20 µL volume was injected and directly analyzed by reverse-phase high-performance liquid-chromatography photo-diode array (HPLC-PDA) using a Thermo Scientific Dionex Ultimate 3000 ultrahigh-performance Liquid chromatography (UHPLC) system with a reverse-phase C1H NMR and 13C NMR) and two-dimensional NMRs (heteronuclear multiple quantum coherence (HMQC), rotating frame Overhauser enhancement spectroscopy (ROESY), and heteronuclear multiple bonded connectivity (HMBC)) were used as needed to elucidate the structure of the compound.One-dimensional NMRs , incubated at 37 °C in a humidified 5% CO2 overnight, and then treated with each compound after serial dilution  for 72 h. After that, 20 μL substrate solution (Promega) was added to each well. The plate was shaken for 5 min and kept in the dark for 10 min. Luminescence was measured using a multimode plate reader . IC50 values were analyzed using GraphPad Prism 5 .Three cancer cell lines , human gastric cancer cell line (AGS), and human leukemia cell line (HL60) and normal cell line (human keratinocyte cell line (HaCaT) were purchased from Korean Cell Line Bank . Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  and AGS cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI1640)  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin-streptomycin-amphotericin B . Human leukemia HL60 cells were cultured in RPMI1640 supplemented with 10% FBS, 1% penicillin-streptomycin-amphotericin B, and L-glutamine (2 mm) . HaCaT cell lines were grown in DMEM supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 μg/mL benzylpenicillin. All cells were maintained at 37 °C in a humidified 5% COStaphylococcus. Aureus CCARM 3640 (MRSA), S. aureus CCARM 3089 (MRSA), S. aureus CCARM 33591(MRSA), S. aureus CCARM 0205 (MSSA), S. aureus CCARM 0204 (MSSA), S. aureus CCARM 0027 (MSSA), S. aureus CCARM 3090 (MRSA), S. aureus CCARM 3634 (MRSA), S. aureus CCARM 3635 (MRSA), Bacillus subtilis ATCC 6633, Enterococcus faecalis 19433, Enterococcus faecalis 19434, Kocuria rhizophilla NBRC 12708, and Micrococcus luteus) and four Gram-negative bacteria  were used to test antibacterial activities of chrysazin and its derivatives. The paper disk diffusion assay on the Mueller–Hinton agar (MHA) plate was carried out according to Clinical Laboratory Standard Institute (CLSI) guidelines and the Kirby–Bauer method [8 colony forming units (CFU)/mL were spread onto MHA plates. Then, 40 µg/disk compounds were placed on the surface of inoculated agar plates using sterile paper disks of 6 mm . Samples were then incubated at 37 °C for 18–20 h. The zone of inhibition diameter was measured in millimeters for each pathogen. Dimethyl sulfoxide (DMSO) was used as a control for the zone of inhibition as all compounds were dissolved in DMSO.Fourteen Gram-positive bacteria , S. aureus CCARM 3089 (MRSA), S. aureus CCARM 33591(MRSA), S. aureus CCARM 0205 (MSSA), S. aureus CCARM 0204 (MSSA), S. aureus CCARM 0027 (MSSA), S. aureus CCARM 3090 (MRSA), S. aureus CCARM 3634 (MRSA), and S. aureus CCARM 3635 (MRSA). They were grown in Mueller–Hinton Broth (MHB) . The broth dilution method was used to determine MIC [8 CFU/mL) and then diluted to 2.5 × 106 CFU/mL in MHB. After knowing the MIC, the MBC test was performed on a fresh MHB medium by inoculating cultured samples containing MIC compounds and experimental strains.The following nine strains were used in the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests: E. coli strain S2 was generated by engineering E. coli BL21 (DE3), which contained a sugar transfer cassette and a sugar methylation cassette. It was cultured and prepared for biotransformation as mentioned in E. coli strain S2 was analyzed by HPLC. The HPLC chromatogram of chrysazin was obtained with its standard retention time (tR) of 21.3 min. Two new peaks of CR and CRM were obtained at tR of 14.9 min and 16.3 min, respectively, with UV absorbance at 420 nm  and 2D NMR , as shown in Biotransformation was carried out through fermentation to collect CR and CRM for structure identification and further biological activity tests. The biotransformation reaction mixture was extracted with a double volume of ethyl acetate. The crude extract was subjected to preparatory-high-pressure liquid chromatography (prep-HPLC) for purification. After several rounds of prep-HPLC, purified compounds were obtained. The purified product was dried by lyophilization, dissolved in 400 µL of deuterated dimethyl sulfoxide, and analyzed by nuclear magnetic resonance (NMR) spectroscopy (700 MHz) including 1D NMR  was consistent with δ 5.67 , in which the anomeric proton coupling constant (J = 1.1 Hz) confirmed that the conjugation of rhamnose moiety had an α-configuration. In addition, with 13C-NMR of CR, the anomeric carbon peak appeared at δ 99.08 ppm, with other peaks appearing between 70 and 80 ppm along with a CH3 peak at 18.35 ppm. In the case of CRM, there was a methylation in the 2′ -OH group of rhamnose in CR, where the OCH3 spectrum was visible in both 1H and 13C NMR at 3.5 ppm and 59.44 ppm, respectively. Furthermore, to confirm the sugar and sugar-O-methylation conjugation, two-dimensional (2D)-NMR analyses such as 1H-13C HSQC, 1H-13C HMBC, 1H-1H COSY, and 1H-1H ROESY experiments were performed. Similarly, in CR, HSQC showed a cross peak illustrating a correlation between the anomeric C-1′ proton (δ 5.67 ppm) and the anomeric carbon (δ 99.00 ppm). Moreover, the C-8 signal appearing at δ 157.18 ppm showed a direct correlation with the observed anomeric proton at δ 5.67 ppm in HMBC (δ 3.71 ppm) and the carbon (δ 80.61 ppm), and HMBC showed a cross peak depicting the correlations between C-2′ (δ 80.62 ppm) and the protons of the methoxy group (δ 3.50 ppm) (O-α-l-rhamnoside) and methylated derivative of CR  produced by E. coli S2 whole-cell biotransformation were new compounds.The  in HMBC . In the .50 ppm) . Results50) values of chrysazin for AGS, Huh7, and HL60 cells were 17.08, 30.53, and 22.24 (μM), respectively. Chrysazin-8-O-α-l-rhamnoside inhibited AGS, Huh7, and HL60 cells with 50% inhibitory concentration (IC50) values of 28.58, 21.28, and 14.68 (μM), respectively. Here, CR showed better anticancer activities in Huh7 and HL60 than chrysazin. In the case of AGS cells, it showed slightly lower anticancer activity. In the case of chrysazin-8-O-α-l-2′-O-methylrhamnoside, it showed higher anticancer activities than chrysazin and CR. The 50% inhibitory concentration (IC50) values of CRM for AGS, Huh7, and HL60 cells were 7.513, 4.467, and 4.540 (μM), respectively. In the case of the HaCaT normal cell line, the IC50 values were >200 μM for chrysazin, CR, and CRM -2,5- diphenyltetrazolium bromide (MTT) colorimetric assay against three different cancer cell lines  and one  and CRM . These cS. aureus subsp.  were prepared at a concentration of 10 mg/mL. All compounds were added to each disk at a final concentration of 40 µg/disk (4 µL). Each disk was placed over Mueller–Hinton agar (MHA) plates spread with bacterial strains. The diameter of the zone of inhibition was measured after 18–20 h. Results of disk diffusion assays revealed that chrysazin did not show any antibacterial activity against 18 different human pathogens tested. However, CR and CRM exhibited antibacterial activities against Gram-positive bacteria s subsp. . These rO-α-l-rhamnoside, and chrysazin-8-O-α-l-2′-O-methylrhamnoside against nine different pathogenic bacteria were determined. MIC values of compound chrysazin, CR, CRM, and erythromycin as a positive control, against nine strains of Gram-positive bacteria S. aureus subsp were determined by the broth dilution method. The assays were performed in 96-well plates in duplicate with Mueller–Hinton broth. As summarized in S. aureus CCARM 0205 (MSSA), S. aureus CCARM 0204 (MRSA), S. aureus CCARM 3640 (MRSA), S. aureus CCARM 3090 (MRSA), S. aureus CCARM 3634 (MRSA), S. aureus CCARM 0027 (MSSA), S. aureus CCARM 3089 (MRSA), S. aureus CCARM 3635 (MRSA), and S. aureus CCARM 33591(MRSA), with MIC values of 7.81–1000 µg/mL. After knowing the MIC value, we further analyzed MBC, the lowest concentration of a compound for killing inoculated bacteria. An MBC test was performed by inoculating cultured samples containing MIC compounds and experimental strains on a fresh MHB medium (Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for chrysazin, chrysazin-8-B medium . MBC valE. coli strain for the sustainable production of different derivatives of chrysazin. Chrysazin-8-O-α-l-rhamnoside and chrysazin-8-O-α-l-2′-O-methylrhamnoside were novel compounds. We also evaluated their activities against three different cancer cell lines . CR and CRM exhibited higher cytotoxicities than their parental compound, chrysazin. More significantly, the evaluation of antibacterial activity revealed promising bioactivities of CR and CRM. This study establishes an engineered microbial platform that can be used to produce novel bioactive compounds. This microbial platform can be further fine-tuned for the production of novel derivatives of other anthraquinones or different compounds. Furthermore, the optimization of bioprocessing parameters and rational engineering of the host using various synthetic biological tools and metabolic engineering can be employed to enhance the production titer.In this study, we successfully engineered an


STING is a direct downstream target of let-7i after brain injury. Furthermore, the intranasal delivery of let-7i agomir can also effectively inhibit STING and is beneficial for inflammation resolution and neuronal survival in a mouse model of pial vessel disruption stroke. Consequently, let-7i agomir is a promising candidate for clinical application as a chemically engineered oligonucleotides-based therapeutic for brain injury.Overcoming the lack of drugs for the treatment of traumatic brain injury (TBI) has long been a major challenge for the pharmaceutical industry. MiRNAs have emerged as potential targets for progress assessment and intervention against TBI. The brain-enriched miRNA let-7i has been proposed as an ideal candidate biomarker for TBI, but its regulatory roles in brain injury remain largely unknown. Here, we find that the expression of let-7i is significantly downregulated in the early stages of a hippocampal stab wound injury. The noninvasive intranasal administration of let-7i agomir significantly improves cognitive function and suppresses neuroinflammation, glial scar formation, and neuronal apoptosis in TBI mice. Mechanically,  Traumatic brain injury (TBI) is one of the leading causes of death and permanent disability worldwide and is a risk factor for developing dementia and neuropsychological diseases . AlthougMicroRNAs (miRNAs) are small single-stranded non-coding RNA molecules (about 22 nucleotides) that govern the processes of RNA silencing and the post-transcriptional regulation of gene expression. As a single miRNA regulates the expression of multiple target genes involved in many cellular processes, manipulating the expression of a single miRNA may affect the entire gene network, thus changing the phenotype of complex diseases. Mounting evidence suggests that miRNAs may serve as potential targets for progress assessment and intervention against TBI because they control a range of physiological and pathological functions, such as cell proliferation, differentiation, apoptosis, and metabolism ,9.Let-7 is a family of evolutionary conserved miRNA that are highly expressed in adult tissues and generally serve as key regulators of developmental processes . Let-7i Here, we aimed to examine whether TBI affects let-7 expression in the brain and to assess whether the restoration of let-7 levels is beneficial for suppressing neuroinflammation and reducing neuronal apoptosis after TBI. Furthermore, the mechanism of let-7 targeting for TBI was also explored.C57BL/6J mice were obtained from the SPF  Biotechnology company and maintained in groups of 3~5 animals on a 12 h light/dark cycle, and they had free access to water and a standard mouse diet. Two-month-old male mice were assigned to undergo TBI or sham surgery. All animal procedures followed the ethical guidelines for the care and use of experimental animals, and all experiments were approved by the Animal Committee of the Institute of Zoology, Chinese Academy of Sciences.Hippocampal stab injury (HSI) was performed as previously described with minor modifications . Two-monPVD strokes were induced as previously described . BrieflyIntranasal administration of agomir-let-7i was conducted as described previously . Agomir-IL-6 forward TACCACTTCACAAGTCGGA, IL-6 reverse AATTGCCATTGCACAACTC; IL-1β forward CCTCAAAGGAAAGAATCTATACCTG, IL-1β reverse CTTGGGATCCACACTCTCC; TNFα forward TTCTCATTCCTGCTTGTGG, TNFα reverse TTGGGAACTTCTCATCCCT; human STING forward CACATCCACTCCAGGTACC, human STING reverse AGAAATAGATGGACAGCAGCA; mouse STING forward CTCATTGTCTACCAAGAACCC, mouse STING reverse TTCTTCCTGACGAATGTGC; let-7a forward ggcgTGAGGTAGTAGGTTGTATA, let-7b forward ggcTGAGGTAGTAGGTTGTGTG, let-7c forward ggccTGAGGTAGTAGGTTGTATG, let-7e forward ggcTGAGGTAGGAGGTTGTATA, let-7f forward ggcggTGAGGTAGTAGATTGTATA, let-7g forward ggcgTGAGGTAGTAGTTTGTACA, let-7i forward ggcTGAGGTAGTAGTTTGTGCT, common miRNA reverse GCAGGGTCCGAGGTATTC.Total RNA was isolated with TRIzol reagent  according to the manufacturer’s instructions. RNA quality was assessed with the Thermo NanoDrop 2000 spectrophotometer to assess 260/280 and 260/230 nm ratios. All RNA samples met a 260/280 ratio >2.0 and 260/230 ratios in the range of 2.0–2.2. cDNA was generated from reverse transcription of 2μg total RNA using a Transcriptor First Strand cDNA Synthesis Kit . cDNA was quantified using the SYBR Green assay, and the relative gene expression levels were calculated against GAPDH or U6 by using the ∆∆Ct method. The primers we used for qRT-PCR were as follows: w/v), 0.3%Triton X-100, and 0.2% sodium azide for 2 h at room temperature. The primary antibodies we used were as follows: anti-Iba1 , anti-GFAP , anti-NeuN . After overnight incubation at 4 °C with primary antibodies and washing with PBS for 30 min, brain sections were incubated with the secondary antibodies conjugated with Alexa Fluor 488 or 594 (1:500). Finally, sections were stained with DAPI and mounted on glass slides with adhesion anti-fade medium. Confocal images were obtained on a ZEISS 710 confocal laser-scanning microscope. Image analyses and quantification were performed using ImageJ software V1.53 .Mice were anesthetized and transcardially perfused with cold phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains were dissected out and postfixed in 4% PFA overnight. Brains were then equilibrated in 30% sucrose and sectioned into segments 40 μm-thick. Brain sections were washed three times (10 min each) with PBS and then blocked in 3% BSA  staining  was performed to detect the neuronal apoptosis. Briefly, hippocampal tissue sections were washed for 10 min with PBS and incubated in 2% BSA and 0.25% triton X-100 for 30 min at RT. Sections were then incubated with 200 μL TUNEL reaction mixture at 37 °C for 1 h, followed by 3 washes with PBS. Subsequently, sections were incubated with anti-NeuN  antibody overnight at 4 °C. Finally, sections were washed for 10 min with PBS 3 times and then incubated with the secondary antibodies conjugated to Alexa Fluor 488  at RT.Brain tissues were lysed with RIPA buffer . Protein samples were separated on 8–12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes . The PVDF membranes were then blocked in TBS-T containing 3% milk and incubated with primary STING antibodies  at 4 °C overnight. PVDF membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) at room temperature for 2 h. Finally, the immunoreactive proteins were treated with enhanced chemiluminescence reagent . The 5200CE Tanon™ Chemi-Image System was used to obtain the images of the blots, and the band intensity of the blots was analyzed using the software ImageJ.STING 3′UTRs were amplified from genomic DNA by PCR and cloned into the dual luciferase reporter vector pmirGLO . Primers were used for the cloning 3′UTRs of STING as follows: mouse STING forward: CTGTGGTCTCCACGATGACTTGA, mouse STING reverse: CACCCAGGTCTCCAACCTTTAAA; human STING forward: CAGTGGTCTCCAAGCCTCTG, human STING reverse: ATGGAACATGACCAGGAGCCA. Mutagenesis of the putative let-7i binding sites on STING CDS or 3′UTR was performed using the Quick-Change II Site-directed Mutagenesis Kit  according to the manufacturer’s protocol. The primers for subcloning the mutated CDS or 3′UTR were as follows: mouse mutant STING forward: CATAatggagtGTTGGATGTTTGGCC, mouse mutant STING reverse: CCAACactccatTATGTCAGCAGTGTT; human mutant STING forward: TCACTGCCTatggagCCTCACG, human mutant STING reverse: TGAGGctccatAGGCAGTGATTATGA. All plasmid constructs were then verified by sequencing. Dual luciferase transfection assays were performed as previously described . A. A10]. AF = 101.2, p < 0.01; Sham vs. HSI(3 dpi), p < 0.01; Sham vs. HSI(7 dpi), p < 0.01; Sham vs. HSI(14 dpi), p = 0.986)  by using qRT-PCR. The expression of let-7i at 3 and 7 dpi was significantly lower than that in the sham group, and recovered to the same level as that in the sham group at 14 dpi (= 0.986) B. Furthehttp://www.targetscan.org, accessed on 1 October 2020). Gene Ontology (GO) term analyses  found that these predicted targets were related to biological functions involving transcription, regulation of transcription, transport, protein phosphorylation, pre-miRNA processing, protein ubiquitination, phosphorylation, and mRNA transport  = 101.2, p < 0.001; 0 h vs. 6h, p < 0.001; 0 h vs. 1 d, p < 0.001; 0 h vs. 2 d, p < 0.001; 0 h vs. 3 d, p = 0.790)  B. These TNF-α, IL-1β, and IL-6 in the hippocampi of agomir-let-7i-treated and scramble-treated mice at day 3 after HSI. Consistent with our expectation, the levels of TNF-α, IL-1β, and IL-6 were significantly elevated in the hippocampi of negative control mice after HSI  = 32.44, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p < 0.05; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.05), IL-1β  = 41.83, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p < 0.05; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.01), and TNFα  = 79.65, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p < 0.01; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.001) after HSI  = 42.42, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p = 0.195; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.001) and microglial branch number was greatly increased  = 32.44, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p = 0.741; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.001) in the agomir-let-7i-treated HSI group  = 43.44, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p < 0.01; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.05)  and Iba1 (a microglia marker) revealed a considerable reduction in the size of the glial scar after agomir-let-7i treatment in comparison to the scramble control, while the sham group did not reveal any glial scar formation in the hippocampus ( < 0.05) E,F. Thes+/NeuN+ cells was significantly decreased in the agomir-let-7i-treated group compared to that in the scramble-treated group  = 41.55, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p < 0.05; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.01)  staining of hippocampal tissue sections from sham, scramble-treated, and agomir-let-7i-treated mice at day 7 post-injury. The number of TUNEL < 0.01) A,B, indiGiven that agomir-let-7i could reduce neuronal apoptosis as well as inflammatory response and glial scar size in the hippocampus, we speculated that agomir-let-7i-treated mice might perform better than scramble-treated mice on learning and memory tests. To test this hypothesis, we conducted the rotarod test and the Barnes maze test to evaluate hippocampal integrity in HSI mice beginning at 15 dpi.F = 173.0, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p < 0.01; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.001)  B, suggesF = 26.36, p < 0.001; Sham vs. HSI + Scramble, p < 0.01; Sham vs. HSI + Agomir-let-7i, p < 0.05; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.01)  D. AltogeSTING (TMEM173) is known to play a pivotal role in responding to pathogenic DNA and self-DNA in the context of neurodegenerative and autoimmune disorders [STING mRNA  = 169.3, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p = 0.803; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.001)  = 35.86, p < 0.001; Sham vs. HSI + Scramble, p < 0.001; Sham vs. HSI + Agomir-let-7i, p = 0.436; HSI + Scramble vs. HSI + Agomir-let-7i, p < 0.01)  D. Moreov < 0.01) E. AltogeF = 159.2, p < 0.001; Sham vs. PVD stroke + Scramble, p < 0.001; Sham vs. PVD stroke + Agomir-let-7i, p < 0.001; PVD stroke + Scramble vs. PVD stroke + Agomir-let-7i, p < 0.001) and protein levels  = 360.4, p < 0.001; Sham vs. PVD stroke + Scramble, p < 0.001; Sham vs. PVD stroke + Agomir-let-7i, p = 0.138; PVD stroke + Scramble vs. PVD stroke + Agomir-let-7i, p < 0.001) of STING were significantly decreased in the cortex of agomir-let-7i-treated PVD stroke mice  A,B. Furtoke mice E,F. TherIn the present study, we found that TBI decreased let-7i and that the intranasal administration of agomir-let-7i reduced brain damage in TBI mice as well as in PVD stroke mice. Agomir-let-7i administration improved cognitive function in brain-injured mice. Mechanistically, agomir-let-7i administration suppressed neuroinflammation, glial scar formation, and neuronal apoptosis after brain injury, suggesting that agomir-let-7i may serve as a potential therapeutic candidate against injury-induced neuroinflammatory and neurodegenerative diseases, such as TBI and PVD stroke.Both TBI and PVD stroke have primary and secondary injury phases regardless of the severity of the insult. The primary injury encompasses mechanical damage to the brain tissues that release signals that activate microglia, astrocytes, and infiltrated peripheral immune cells to initiate the secondary injury, including inflammation, excitotoxicity, mitochondrial impairment, and neuronal cell death. The secondary injury phase evolves over minutes to days to months after the primary injury, suggesting that early interventions may ameliorate brain damage and stimulate neural regeneration and repair . The maiHuman TBI is a sophisticated disease process, and the diversity in the extent of injury as well as in pathoanatomical subtypes means that different patients are likely to experience different courses and outcomes of TBI. In addition, it is still difficult to generate animal models that can fully recapitulate all the pathophysiological aspects of human TBI. Let-7i has been considered as a promising serum biomarker for stroke and blast-induced TBI. For example, the expression of let-7i is upregulated in blood samples collected from patients with post-stroke cognitive impairment compared with patients with post-stroke cognitive normality . SimilarSTING) was a downstream target of let-7i in the brain, and the intranasal administration of agomir-let-7i could suppress the upregulation of STING, neuroinflammation, and glial scar formation in both TBI and PVD stroke mice. These findings are consistent with previous work that identified STING as a key regulator of inflammation in both rodents and human brain organoids and found that the inactivation of STING attenuates inflammation [Neuroinflammation plays an essential role in the pathophysiology of TBI and stroke. Although a low degree of neuroinflammation is initially beneficial for debris clearance and repair, a high degree of neuroinflammation elicits secondary injury that leads to chronic inflammation and neurodegeneration . Mechaniammation ,32,42,43ammation . Althougammation . Our datIn summary, we found that brain-enriched miRNA let-7i was significantly downregulated at the early stages of TBI in mouse brains. STING was a direct downstream target of let-7i, and agomir-let-7i could protect brain tissue from neuroinflammation, glial scar formation, and neural cell death after brain injury. Overall, our data suggest that the intranasal administration of agomir-let-7i is a potential therapeutic strategy for neurotrauma and PVD stroke.


The rumen contains a complex microbial ecosystem that degrades plant materials, such as cellulose and hemicellulose. We herein reconstructed 146 nonredundant, rumen-specific metagenome-assembled genomes (MAGs), with ≥50% completeness and <10% contamination, from cattle in Japan. The majority of MAGs were potentially novel strains, encoding various enzymes related to plant biomass degradation and volatile fatty acid production. The MAGs identified in the present study may be valuable resources to enhance the resolution of future taxonomical and functional studies based on metagenomes and metatranscriptomes. This study was the first to use JB (arXiv.https://arxiv.org/abs/1303.3997) to the bovine reference genome ARS-UCD1.2/bosTau9. Filtered reads were assembled in SPAdes version 3.13.0 (arXiv.https://arxiv.org/abs/1303.3997). We binned MAGs with contigs using MetaBAT2 version 2.15  to prevent arbitrary mapping between similar genomes  with the following parameters: -m relative_abundance --min-read-percent-identity 0.95 --min-read-aligned-percent 0.75. We taxonomically classified RUG1ANI99% MAGs in GTDB-tk version 2.1.1  with GTDB release 207. We then built a phylogenetic tree in PhyloPhlAn version 3.0.60 , including Hungate1000 (strains (<99%) using the ANI outputs by dRep and GTDB-tk.Filtered reads were mapped against RUG1n 3.0.60 . To elucgate1000  using dRANI99% MAGs with Prodigal version 2.6.3 (Genomes (KEGG) database through GhostKOALA ( Carbohydrate-active enzyme (CAZyme) families, which are associated with cell wall degradation and essential for efficient lignocellulose processing in ruminants, were annotated using dbCAN2 (–5 to search cohesin (PF00963) and dockerin (PF00404) domains. We used PULpy  in 5,414 public Bacteroidetes genomes using PULpy. bioRxiv.https://doi.org/10.1101/421024) to predict polysaccharide utilization loci (PUL), linked gene clusters that encode the cell envelope-associated enzymes required for sensing, binding, and degrading polysaccharide substrates . Following dereplication at 99% ANI, we generated 146 nonredundant RUG13.2 kb . A compaectively . TherefoANI95% MAGs, we identified 32 MAGs that were present (relative abundance >0) in >90% of the tested cattle (n=20), suggesting that they are core rumen bacteria in Japan , Actinobacteriota (10 MAGs), Firmicutes (9 MAGs), Verrucomicrobiota (7 MAGs), Spirochaetota (4 MAGs), Patescibacteria (3 MAGs), Proteobacteria (2 MAGs), Methanobacteriota (2 MAGs), Planctomycetota (1 MAG), Synergistota (1 MAG), Desulfobacterota_I (1 MAG), and Elusimicrobiota (1 MAG). In Bacteroidota MAGs, 10 RUG1ANI99% MAGs were classified as Prevotella, which is the most abundant bacterial genus in the rumen  with at least one gene related to acetate production . Firmicutes_C RUG1ANI99%These genes were absent from  MAGs. Rumen propionate is produced via the succinate and acrylate pathways . Prevotella  and GH77 (4-α-glucanotransferase), respectively, both of which are involved in starch degradation , GH3 (β-glucosidase), GH5 , GH13 (α-amylase), GH26 , GH32 (invertase), GH36 , GH43 (β-xylosidase), GH73 (lysozyme), GH94 (cellobiose phosphorylase), and GH97 (α-glucosidase).Among 146 RUG1radation . Further CAZymes , which iANI99% MAGs. The proteins containing cohesin and dockerin domains are summarized in ANI99% MAGs, including 2 Ruminococcus and 11 Ruminococcus_E MAGs. Sixteen dockerin-containing proteins carried CAZyme domains, mostly including GH families containing α-amylase . Among the 25 RUG1ANI99% MAGs, 13 had both cohesin- and dockerin-containing proteins, while 9 had only dockerin-containing proteins. Ruminococcus albus 8, which degrades cellulosic substrates, harbored no cohesion-containing protein. Therefore, we cannot rule out the possibility that RUG1ANI99% MAGs, which had no cohesion- and some dockerin-containing proteins, used an alternative mechanism for the immobilization of dockerin-containing enzymes onto carbohydrates, similar to Ruminococcus albus 8 . Ninety-eight and 80 PULs were identified in Cryptobacteroides MAG and Prevotella MAGs, respectively, and contained various CAZymes. Overall, the present results suggest that Cryptobacteroides and Prevotella are important for rumen function  in cattle in Japan.In  domains . The mosIn summary, we reconstructed 146 rumen-specific MAGs from cattle in Japan, with many being potentially novel. These MAGs are valuable resources for enhancing the resolution of future metagenome- and metatranscriptomic-based taxonomical and functional studies.Microbes Environ 37: ME22039.Sato, Y., Takebe, H., Oishi, K., Yasuda, J., Kumagai, H., Hirooka, H., and Yoshida, T. (2022) Identification of 146 Metagenome-assembled Genomes from the Rumen Microbiome of Cattle in Japan. https://doi.org/10.1264/jsme2.ME22039Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7


Correction: Immun Ageing 19, 57 (2022)https://doi.org/10.1186/s12979-022-00310-yFollowing publication of the original article , the autThe incorrect author name is: Jéssica S. de GuedesThe correct author name is: Jéssica de S. GuedesThe author group has been updated above and the original article  has been


ENDOGLIN (ENG) gene. Here, we generated induced pluripotent stem cells (hiPSCs) from a patient with rare mosaic HHT1 with tissues containing both mutant (ENGc.1678C>T) and normal cells, enabling derivation of isogenic diseased and healthy hiPSCs, respectively. We showed reduced ENG expression in HHT1 endothelial cells (HHT1-hiPSC-ECs), reflecting haploinsufficiency. HHT1c.1678C>T-hiPSC-ECs and the healthy isogenic control behaved similarly in two-dimensional (2D) culture, forming functionally indistinguishable vascular networks. However, when grown in 3D organ-on-chip devices under microfluidic flow, lumenized vessels formed in which defective vascular organization was evident: interaction between inner ECs and surrounding pericytes was decreased, and there was evidence for vascular leakage. Organs on chip thus revealed features of HHT in hiPSC-derived blood vessels that were not evident in conventional 2D assays.Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by weak blood vessels. HHT1 is caused by mutations in the   •Vessels from isogenic hiPSCs from HHT1 patients compared•HHT1-hiPSC-ECs show defective vascular organization in 3D microfluidic chips•HHT1-hiPSC-ECs show defective EC-pericyte interaction In this article, Orlova and colleagues describe 3D vessels on chip (VoCs) composed of hiPSC-ECs from a patient with hereditary hemorrhagic telangiectasia (HHT1). HHT1-hiPSC-ECs and healthy isogenic controls behaved similarly in 2D culture, but defective vascular organization and reduced pericyte coverage were evident in HHT1-hiPSC-ECs in VoCs. The model is thus a valuable tool for mechanistic studies and future drug discovery. ENG; HHT1), Activin receptor like kinase-1  or SMAD4 (HHT3), genes that mediate signaling by transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) in vascular endothelial cells (ECs) (in vitro makes them unsuitable as a renewable source of ECs for reproducibly modeling the disease in humans and for drug discovery.Hereditary hemorrhagic telangiectasia (HHT) is an inherited genetic disorder caused by autosomal dominant mutations in Endoglin (ls (ECs) . Phenotyls (ECs) . These als (ECs) . More sels (ECs) . To datels (ECs) . Medicalls (ECs) , thalidols (ECs) , itraconls (ECs) , and othls (ECs) . Geneticls (ECs) . Attemptls (ECs) . Blood ols (ECs) , but thein vitro and (2) investigating defective endothelial-pericyte interactions.In the present study, we aimed to establish an efficient and scalable system that would recapitulate the formation of defective blood vessels in patients with HHT, based on patient-derived human induced pluripotent stem cells (HHT1-hiPSCs). We hypothesized that HHT1-hiPSCs might be useful for (1) identifying mechanisms underlying disease predisposition and modeling clinical features of HHT1 ENG (NM_001114753.2 (ENG):c.1678C>T; p.(Gln560∗)), which causes ENG haploinsufficiency (c.1678C>T and HHT1WT) (c.1678C>T-hiPSC-ECs compared with HHT1WT-hiPSC-ECs (ID1 expression was significantly upregulated in HHT1c.1678C>T-hiPSC-ECs after 2 h of TGF-β treatment (hiPSC lines were generated from somatic tissue from a patient with HHT1 with a heterozygous nonsense mutation in ficiency . The pat HHT1WT) D–S1G. HH HHT1WT) ; 2014b. iPSC-ECs A and 1B.iPSC-ECs B. ENG haiPSC-ECs A. HHT1-hreatment B.Figure c.1678C>T-hiPSC-ECs  C. BarrieiPSC-ECs C and S2DiPSC-ECs C and S2DiPSC-ECs E.in vitro was examined, as described previously .Finally, the ability to form 2D vascular networks eviously , 2014b. eviously F. Quantibranches G as wellc.1678C>T-hiPSC-ECs compared with HHT1WT-hiPSC-ECs  model was then examined A. PrimariPSC-ECs B and 2C,iPSC-ECs A. QuantiiPSC-ECs C and S3Dimilarly B. Furthee nuclei E and S3Fe nuclei . Notablyc.1678C>T-hiPSC-ECs compared with HHT1WT-hiPSC-ECs.Junctional integrity was examined by immunostaining of the microvascular networks with VEC and ZO1 D and 2E.c.1678C>T-hiPSC-ECs showed reduced pericyte coverage compared with HHT1WT-hiPSC-ECs   C–S4E.Figc.1678C>T-hiPSC-ECs compared with HHT1WT-hiPSC-ECs  was added into the medium channel of the organ-on-chip device, and real-time videos of vascular segments pre-stained using fluorescent agglutinin were made . QuantifiPSC-ECs B and 4C.in vitro model for the genetic vascular disorder HHT using hiPSCs derived from patients with mutations in the ENG gene (HHT1). The results showed that we likely captured the direct effects of reduced ENG protein on the EC surface without compensation or adaption mechanisms that normally occur in vivo, notably in mutant mice  was used to knock down onic ECs . Completin vivo  . The generation of the lines was approved by the Leiden University ethics committee under the P13.080 “Parapluprotocol: hiPSC.” Patient samples, fibroblasts from skin biopsies, and erythroblasts isolated from peripheral blood were used for reprogramming. Reprogramming with episomal vectors was done as described, except that a newer generation of vectors without ere used . hiPSCs ere used . KaryotyRA-FISH) , and pluOne-way ANOVA and non-parametric Student’s t test for unpaired measurements were applied as appropriate to test for differences in means between the groups. Detailed statistics are indicated in each figure legend. Data are expressed and plotted as the mean ± SD. Statistical significance is indicated in each figure legend. Statistical analysis was performed with GraphPad Prism 9.0.2.V.V.O., designed the research, established functional assays, performed experiments, and wrote the manuscript; D.M.N. and A.C., performed experiments in 3D vascular chips, did imaging, and performed quantification; X.C., performed EC differentiation; C.F., performed reprogramming experiments; F.v.d.H., conducted EC differentiation and isolation and FACS; F.L., assisted with quantification of microfluidic experiments; C.J.J.W., R.J.S., and H.-J.M., provided HHT patient samples; J.K.P.v.A., conducted genetic analysis; P.t.D. and F.L. helped analyze the data; C.L.M., designed the research and wrote the manuscript.The authors declare no competing interests.


This study inventively combines epidermal growth factor receptor (EGFR) expression of the primary lesion and standardized uptake value (SUV) of positron emission tomography and computed tomography (PET/CT) to predict the prognosis of nasopharyngeal carcinoma (NPC). This study aimed to evaluate the predictive efficacy of maximum standard uptake value (SUVmax) and EGFR for treatment failure in patients with NPC.18F-FDG PET/CT of 313 patients with NPC. Time-dependent receiver operator characteristics was used for analyzing results and selecting the optimal cutoff values. Cox regression was used to screen out multiple risk factors. Cumulative survival rate was calculated by Kaplan–Meier.This retrospective study reviewed the results of EGFR expression and pretreatment p = 0.0083), locoregional relapse-free survival (LRRFS) (p = 0.0077), distant metastasis-free survival (DMFS) (p = 0.013), and progression-free survival (PFS) (p = 0.0018) among the four groups. Patients in the EGFR-positive and SUVmax-T > 8.5 group had the worst survival, while patients in the EGFR-negative and SUVmax-T ≤ 8.5 group had the best prognosis. Subsequently, patients with only positive EGFR expression or high SUVmax-T were classified as the middle-risk group. There were also a significant difference in 3-year overall survival among the three risk groups (p = 0.034). SUVmax-T was associated with regional recurrence-free survival and LRRFS in multivariate analysis, whereas EGFR was an independent prognostic factor for LRRFS, DMFS, and PFS.The selected cutoff value of SUVmax-T was 8.5. The patients were categorized into four groups according to EGFR expression and SUVmax-T. There were significant differences in the 3-year local recurrence-free survival (LRFS) (The combination of SUVmax-T and EGFR expression can refine prognosis and indicate clinical therapy.The online version contains supplementary material available at 10.1186/s13014-023-02231-6. Nasopharyngeal carcinoma (NPC) is a highly aggressive malignant tumor believed to arise from nasopharyngeal epithelial cells . Accordi18F-fluorodeoxyglucose positron emission tomography and computed tomography (18F-FDG PET/CT) have been frequently used in pretreatment diagnostic evaluation and post-treatment monitoring because of their unique capability to image metabolically active lesions [The  weight)]. SUVmax-Radiotherapy dose and target volume delineation were performed according to the recommendations (Radiation Therapy Oncology Group) , 23. ThePatients with stage I were treated with radiotherapy alone, while patients with stage II-IV received radiotherapy combined with chemotherapy .After RT, follow-up was conducted once every 3 months for the first 2 years, once every 6 months in years 3 to 5, and annually thereafter. The final follow-up date was March 2022. Study endpoints included local recurrence-free survival (LRFS), regional recurrence-free survival (RRFS), locoregional relapse-free survival (LRRFS), distant metastasis-free survival (DMFS), progression-free survival (PFS), and OS.P-values < 0.05 indicated statistical significance.Uses IBM SPSS statistical software version 22.0 and R software version 4.0.5 for the statistical analysis. The optimal cutoff values was decided by Time-dependent receiver operator characteristic (ROC) analysis . Kaplan–Meier methods were used to compute the survival analyses. Between-group differences in survival outcomes were assessed using log-rank tests. Cox regression was used to screen out multiple risk factors. All tests were two-tailed, and The clinical characteristics of all eligible patients are summarized in Table P = 0.075), 97.9% vs. 93.2% (P = 0.084), 94.7% vs. 87.6% (P = 0.032), 97.0% vs. 87.4% (P = 0.018), 91.8% vs. 76.7% (P = 0.0017), and 96.5% vs. 90.8% (P = 0.058), respectively .Among 1877 patients with NPC with IHC examination for primary lesions, EGFR was detected in 73.36% (1377 patients), and the proportion of patients showing negligible intensity (negative) of EGFR staining was 26.64% (500 patients). For the enrolled 313 patients, 241 (77.00%) showed positive EGFR expression, while 72 (23.00%) showed negligible expression (negative). The 3-year LRFS, RRFS, LRRFS, DMFS, PFS, and OS rates in the EGFR-negative group vs. EGFR-positive group were 96.9% vs. 92.2% (p = 0.0038), 96.3% vs. 95.3% (p = 0.21), 93.8% vs. 84.2% (p = 0.0063), 93.5% vs. 86.5% (p = 0.022), 85.8% vs. 75.5% (p = 0.018), and 94.4% vs. 93.5 (p = 0.066), respectively .The mean SUVmax-T was 10.21 ± 5.59 , and the mean SUVmax-N was 7.53 ± 5.55 . To further evaluate the prognostic value of SUVmax, time-dependent ROC analysis was used to determine the optimal cutoff values based on the 3-year survival outcome. The optimal cutoff value of SUVmax-T was 8.5 based on the 3-year LRFS , LRRFS (p = 0.0077), DMFS (p = 0.013), and PFS (p = 0.0018) .To better predict the prognosis of NPC, patients were divided into the following four groups based on SUVmax-T and EGFR expression: (a) EGFR negative and low SUVmax-T, (b) EGFR negative and high SUVmax-T, (c) EGFR positive and low SUVmax-T, and (d) EGFR positive and high SUVmax-T. There were obviously statistical difference in 3-year LRFS (18) Fig.  among thp = 0.0029), LRRFS (p = 0.0026), DMFS (p = 0.005), and PFS (p = 0.00073), the 3-year OS also showed significant difference among 3 risk groups .Figure p = 0.005) and LRRFS . Moreover, multivariable survival analysis revealed that EGFR was an independent prognostic factor for LRRFS , DMFS , and PFS .Six variables  were included in the univariate analysis for the six clinical endpoints, and the results are summarized in Additional file The most important prognostic factor for NPC is TNM clinical stage. However, there is considerable variability in outcomes among patients with the same TNM stage receiving the same treatment . Recent PET/CT is functional imaging, which is different from morphology and structure imaging. The parameters of PET can be used to characterize the burden of metabolically active lesions and biological aggressiveness in malignancies. SUV, TLG, and MTV are parameters that have been correlated with survival outcome –16. SUVmIn 2008, a retrospective study have shown SUVmax may predict DFS in NPC treated with CCRT and more aggressive treatment should be given to patients with higher SUVmax . A prospSeveral meta-analyses have shown that EGFR overexpression is significantly associated with poor OS and DFS. Thus, EGFR may serve as a potential prognostic predictor of NPC , 37, 38.p = 0.0083), LRRFS (p = 0.0077), DMFS (p = 0.013), and PFS (p = 0.0018) among the four groups. The K–M curve revealed that patients in the EGFR-positive and SUVmax-T > 8.5 group had the worst survival, while patients in the EGFR-negative and SUVmax-T ≤ 8.5 group had the best prognosis. For patients in the EGFR-negative + SUVmax-T > 8.5 group and EGFR-positive + SUVmax-T ≤ 8.5 group, the cumulative survival curves were extremely close. This demonstrated that EGFR expression and high SUVmax-T may be adverse prognostic factors for NPC. Therefore, we further divided patients into three groups. Patients with positive EGFR expression or high SUVmax-T levels were defined as the middle-risk group. The 3-year OS also showed a significant difference among the three risk groups (p = 0.034). Moreover, SUVmax-T was associated with LRFS and LRRFS in multivariate analysis, whereas EGFR was an independent prognostic factor for LRRFS, DMFS, and PFS.To better predict the clinical outcomes of de novo NPC, we combined functional imaging data with molecular pathological data. Patients were categorized into four groups based on EGFR expression (negative or positive) and SUVmax-T (≤ 8.5 or > 8.5). There were significant differences in 3-year LRFS ; B regional recurrence-free survival (RRFS); C locoregional relapse-free survival (LRRFS); D distant metastasis-free survival (DMFS); E progression-free survival (PFS); F overall survival (OS). Fig. S3: Kaplan-Meier curves in the low SUVmax-T (≤8.5) group and the high SUVmax-T (>8.5) group. A local recurrence-free survival (LRFS); B regional recurrence-free survival (RRFS); C locoregional relapse-free survival (LRRFS); D distant metastasis-free survival (DMFS); E progression-free survival (PFS); F overall survival (OS).


Vigna unguiculata (lobia) production remains poorly understood. Thus, we aimed to isolate and characterize the soil microbes from the rhizosphere and develop novel microbial consortia for enhancing lobia production. Fifty bacterial strains were isolated from the rhizosphere soil samples of lobia. Finally, five effective strains  were identified and molecularly characterized by 16 S rDNA gene amplification. All selected strains showed positive plant growth promoting (PGP) properties in broth culture. Based on morphological, biochemical, and plant growth promoting activities, five effective isolated strains and two collected strains (Azospirillum brasilense MTCC-4037 and Paenibacillus polymyxa BHUPSB17) were selected. The pot trials were conducted with seed inoculations of lobia (Vigna unguiculata) var. Kashi Kanchan with thirty treatments and three replications. The treatment combination T3 (Pseudomonas sp. IESDJP-V2), T14 (Pseudomonas sp. IESDJP-V2 + A. brasilense), T26 (Pseudomonas sp. IESDJP-V1+ B. cereus IESDJP-V4 + P. polymyxa) and T27 (IESDJP-V1+ IESDJP-V5+ A. brasilense) were recorded for enhancing plant growth attributes, yield, nutritional content like protein, total sugar, flavonoid and soil properties as compared to control and others. The effective treatments T3 (Pseudomonas sp.), T14 (Pseudomonas sp. IESDJP-V2 + A. brasilense), T26 (Pseudomonas sp. IESDJP-V1+ B. cereus IESDJP-V4 + P. polymyxa) and T27 (IESDJP-V1+ IESDJP-V5+ A. brasilense) recorded as potential PGPR consortium for lobia production. The treatment of single (Pseudomonas sp.), duel (IESDJP-V2 + A. brasilense) and triple combination (IESDJP-V1+ IESDJP-V4 + P. polymyxa) and (IESDJP-V1+ IESDJP-V5+ A. brasilense) can be further used for developing effective indigenous consortium for lobia production under sustainable farming practices. These PGPR bio-inoculant will be cost-effective, environment-friendly and socially acceptable.The rhizosphere microbes play a key role in plant nutrition and health. However, the interaction of beneficial microbes and  The gThe application of microbial consortium or inoculum in single, dual, triple, tetra, penta and hexa, and more combinations are widely used for increasing sustainable agricultural productivity as well as enhancing soil fertility and health ,8. PGPR Vigna unguiculata L.) is an important seed legume crop that is broadly used as green vegetables in India and other countries of World , 10.13039/501100021098Design and Innovation Centre, BHU and Indian Institute of Technology (IIT-BHU) Varanasi [DIC-BHU/Project S-23 Approval/2016-17/693].Professor Jay Prakash Verma was supported by Data associated with this study has been deposited at NCBI-GenBank and got accession number of Pseudomonas sp. IESDJP-V1 (MH362754) and Pseudomonas sp. IESDJP-V2 (MH362755), S. marcescens IESDJP-V3 (MH362756), B. cereus IESDJP-V4 (MH362757), Ochrobactrum sp. IESDJP-V5 (MH362758).The authors declare no competing interests.No additional information is available for this paper.


Pseudanabaena spp. are filamentous cyanobacteria widely distributed in temperate lakes. Though infrequent, they can form harmful algal blooms. Here, we present a high-quality metagenome-assembled genome of a Pseudanabaena sp. from a toxic, crimson cyanobacterial bloom in Lake Salubria, NY. Pseudanabaena spp. are filamentous, nonheterocystous cyanobacteria widely distributed in temperate lakes . In spring 2020, an algal bloom turned the lake a vivid crimson, concerning the community . Grab saN50 = 5,395 bp).Filaments were filtered onto a 5-μm polycarbonate filter from which genomic DNA was extracted using standard phenol-chloroform methods (De novo assembly was conducted using Unicycler (v0.4.9b) in normal mode  . Illuminmal mode . CheckM mal mode . GTDB-Tkmal mode . The genmal mode . The 16SPseudanabaena sp. Salubria-1, consisting of 107 contigs with a total length of 7,138,199 bp  and a GC content of 41.93%. The sequencing depth was ~191-fold. The genome was estimated at 99.1% complete with 3.3% contamination. The closest taxonomic placement (91.9% average nucleotide identity [ANI]) was to Pseudanabaena sp. strain UWO311, a red strain isolated from Dickson Lake, Ontario, Canada (Pseudanabaena/Limnothrix group (We recovered a MAG of bp N50 = 5,348 bp JALQCR000000000. Reads are deposited in the NCBI Sequence Read Archive under accession numbers SRX15003418 and SRX15003417.The metagenome-assembled genome is deposited at DDBJ/ENA/GenBank under accession number


In this study, we aimed to investigate the effect of p62 on angiogenesis and microRNA (miRNA) expression profiles in acute myeloid leukemia (AML) exosomes.An Exiqon v19.0 microRNA MicroArray was used to profile miRNAs in exosomes derived from parental and p62-knockdown U937 cells. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to predict the biological functions and potential mechanisms of differentially expressed miRNAs in AML exosomes. Endothelial cell tube formation assays using human umbilical vein endothelial cells (HUVECs) were performed to investigate the effect of AML exosomes on angiogenesis.P < 0.05). GO analysis indicated that miRNAs were most enriched in the intercellular pathways. Biological process analysis revealed that 1460 biological processes were associated with downregulated transcripts, including 19 pathways related to vesicles, and 1,515 pathways were upregulated, including 8 pathways related to vesicles. Molecular function analysis indicated that protein binding, transcription regulator activity, and DNA-binding transcription factor activity were enriched (P < 0.05). Pathway analysis indicated that 84 pathways corresponded to upregulated transcripts, and 55 pathways corresponded to downregulated transcripts (P < 0.05). We also found that exosomes derived from U937 cells promoted angiogenesis in HUVECs.We demonstrated that 2,080 miRNAs were expressed in exosomes derived from our cultured cell samples, of which 215 and 208 miRNAs were upregulated and downregulated, respectively, in p62-knockdown U937 cells (fold change ≥ 2, Our data suggest that exosomal miRNAs may play important roles in the pathogenesis of AML, which may be treated by p62 knockdown with exosomal miRNAs to inhibit angiogenesis. Acute myeloid leukemia (AML) is a fatal hematological malignancy with high recurrence rate. For patients receiving the most intensive treatment, the overall 5-year survival rate remains below 50%. For the remaining patients, the prognosis is even worse . AML is Exosomes are nanometer-scale extracellular vesicles containing many microRNAs (miRNAs) that are secreted from cells in both normal and pathological conditions . It has In this study, we constructed a miRCURYTM LNA Array (v.19.0) of miRNAs in exosomes derived from AML cells after p62 knockdown. The miRNAs in exosomes were analyzed by identifying signature miRNAs. We then investigated angiogenesis in human umbilical vein endothelial cells (HUVECs) exposed to exosomes derived from parental U937 cells, p62-knockdown U937 cells, or control cells. The data from these studies may shed light on the relationship between exosomal miRNAs and AML, further enhancing our understanding of AML progression.Our study may aid the development of potential biomarkers for the diagnosis and prognosis of AML progression.SQSTM1 gene (LV-SQSTM1-RNAi) and an empty recombinant adenovirus vector (Hu6-MCS-CMV-EGFP) were constructed. U937 cells were placed in a six-well plate. Polybrene  was used for the transfection. The transfection system included 1.8 mL RPMI-1640 with 10% fetal bovine serum, 10 µL LV-SQSTM1-RNAi or Hu6-MCS-CMV-EGFP, and 0.9 µL polybrene. After transfection for 24 h, the cell suspension was collected and centrifuged at 800 rpm for 5 min, the supernatant was discarded, and two mL of RPMI-1640 with 10% fetal bovine serum was added. After transfection for 48 h, fluorescence was observed. After culturing, 5 µg/mL puromycin was added, and the cells were screened for 15 days. After selecting the surviving cells, cell lines with clonal stability were cryopreserved and characterized through RT–qPCR and western blotting.The human acute monocytic leukemia cell line U937 was purchased from the Bena Culture Collection  and stored in our laboratory. A recombinant lentivirus vector-mediated 4 cells/well in 96-well plates and allowed to grow for 12, 24, and 48 h. Next, 10 µL CCK-8 solution  was added to the cell suspension and incubated for 2 h. Absorbance was measured using a spectrophotometer at 450 nm. The experiment was repeated at least three times.U937 cells were plated at a density of 3–5 × 10Flow cytometry analysis using an Annexin V-FITC/PI detection kit was used to compare the apoptosis rate of p62-control and p62 knockdown U937 cells. After 48 h of incubation, the cells were washed with phosphate-buffered saline and resuspended in 400 µL of 1 × binding buffer. Thereafter, 5 µL Annexin V-FITC and 5 µL PI were added to the mixture and stained in the dark for 15 min at room temperature. Apoptosis was detected using flow cytometry  immediately after staining.P62-siRNA coated with lentivirus interfered with U937 cells to downregulate the expression of p62, with the empty virus vector used as a control. Two groups of cells were used as follows: p62-knockdown U937 cells and controls. The two groups of cells were cultured for 48 h in serum-free media. The supernatant was collected for exosome extraction via ultracentrifugation. Exosome shape and size were observed using electron microscopy, and exosomal markers were detected using western blot analysis.Total protein was extracted from U937 cells that had or had not been transfected with the p62-encoding gene using radioimmunoprecipitation assay lysis buffer . The protein concentration was determined using the BCA method. Equal amounts of protein samples were added to each well, separated using 10% SDS-PAGE, and transferred to a polyvinylidene chloride transfer membrane . The membrane was blocked with 5% skimmed milk for 2 h. It was then washed with TBST and incubated with primary antibodies against p62, TSG101, CD63, CD9, calnexin, and GAPDH  overnight at 4 °C. Thereafter, the membrane was incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h after washing three times with TBST. An enhanced chemiluminescence substrate (Thermo Fisher Scientific) was used to detect the protein bands. Image Lab software was used to detect and analyze the density of each band .TRIzol  was used to extract total RNA. A NanoDrop spectrophotometer  was used to measure RNA quality and quantity. RNA integrity was assessed using gel electrophoresis. After quality control, miRNA labeling was performed according to the instructions of the miRCURY™ Hy3™/Hy5™ Power Labeling Kit . First, 1 µL RNA in 2 µL water was mixed with 1 µL CIP buffer and CIP . The mixture was then incubated at 37 °C for 30 min. The mixture was incubated at 95 °C for 5 min to stop the reaction. Then, 3 µL labeling buffer, 1.5 µL fluorescent label (Hy3TM), 2 µL dimethyl sulfoxide, and 2 µL labeling enzyme were added. The mixture was then incubated for 1 h at 16 °C, followed by 15 min at 65 °C to terminate the reaction. Hy3-labeled samples were hybridized on the miRCURYTM LNA array  according to the manufacturer’s instructions. A total of 25 µL Hy3™-labeled samples and 25 µL hybridization buffer were denatured at 95 °C for 2 min and then incubated on ice for 2 min. The hybridization system  was used with the microarray set at 56 °C for 16 to 20 h. After hybridization, the slides were washed several times using a washing buffer kit . Finally, an Axon GenePix 4000 B microarray scanner  was used to scan the slides.P value. Finally, hierarchical clustering was used to show the different miRNA expression profiles between the samples.GenePix Pro 6.0 software  was used to extract data by analyzing the imported scanned images. The samples were chosen to calculate normalization factors if the replicated miRNAs were averaged and for miRNAs with intensities ≥30. Median normalization was used to normalize the data. Normalized data = (foreground background)/median; the median was the 50% quantile of miRNA intensity, which was larger than 30 in all samples after background correction. After normalization, the miRNAs with significant differences between the two groups were determined according to the fold change and http://www.targetscan.org/) and mirdbV5 (http://mirdb.org/) are online sites for miRNA target gene prediction (TargetScan7.1 (ediction . In our http://www.geneontology.org) and KEGG (http://www.genome.ad.jp/kegg/) databases to study the potential organisms and signaling pathways of differentially expressed miRNAs. Differences were considered statistically significant at P < 0.05.We used the GO , seeded at 3 × 104 cells per well. Tubules were photographed using phase microscopy after incubation for 0, 3, and 6 h at 37 °C with 5% CO2.t-test, and statistical significance was set at P < 0.05.We used GraphPad Prism 6 to corroborate the statistical significance of the data for all graphs in this study. Values are presented as the means ± standard deviation. Differences between groups were analyzed using Student’s To generate p62-knockdown U937 cells, we used a lentivirus to transfect cells and observed the transfection efficiency by fluorescence microscopy. We then conducted RT–qPCR analysis to determine the expression levels of the autophagy gene encoding p62 –1B and wP < 0.05).We observed exosome formation using electron microscopy after ultracentrifugation. Electron microscopy revealed exosomes as vesicles with a double-layer membrane structure   –4F, biol < 0.05)  and 6A.  < 0.05) . As for  < 0.05) . The miR < 0.05)  and 6A.http://www.targetscan.org/) and mirdbV5 (http://mirdb.org/) were employed to predict the potential target genes of miRNAs. We found that 2,018 genes co-expressed with upregulated miRNAs and 2,749 genes co-expressed with downregulated miRNAs in the two databases . Among the three groups of HUVECs+Exo(U937), HUVECs+Exo(p62-con) and HUVECs+Exo(p62-), the HUVECs+Exo(p62-) group grew slower than the other two groups (p < 0.05). In summary, the above results indicated that the fastest angiogenesis occurred in the presence of exosomes from U937 cells; the slowest angiogenesis occurred in the presence of exosomes from p62-knockdown U937 cells.HUVECs were inoculated in Matrigel and incubated with AML exosomes for a certain period of time to determine whether exosomes from AML cells could induce HUVEC tubular differentiation nificant . Microscnificant –7C. By cAlthough advances have been made in AML supportive care, prognostic risk stratification, and established therapies, patients with AML have poor long-term prognosis . The ideAccumulating evidence indicates that exosomes in tumors are oncogenic. Crosstalk between bone marrow tumors and endothelial cells can affect tumor progression in hematological tumors, and exosomes containing miRNAs are crucial in bone marrow angiogenesis promotion in hematological tumors. By delivering miR-365, exosomes can mediate the horizontal transfer of drug resistance in chronic myeloid leukemia cells . K562 ceP < 0.05). We used microarray technology to study the expression patterns of exosomal miRNAs derived from two U937 cell lines to further explore the relationship between exosomal miRNAs and p62. We identified 2,080 miRNAs, including 215 upregulated and 208 downregulated miRNAs in p62-knockdown U937 cells. To further validate microarray analysis results, we performed RT–qPCR to validate the downregulated expression of miR-3064-3p and miR-339-5p in the same series of samples. Our future studies will involve identification of other miRNAs whose expression was the highest in our microarray results. The RT–qPCR results were consistent with those obtained from microarray analysis. In addition, KEGG pathway analysis revealed that 84 pathways corresponded to upregulated transcripts, and 55 pathways corresponded to downregulated transcripts. For the downregulated transcripts, the most affected pathway was the “TNF signaling pathway” (Pathway ID: hsa04668), followed by the “MAPK pathway” (Pathway ID: hsa04010). As for upregulated transcripts, the “PI3K–Akt signaling pathway” (Pathway ID: hsa04151) was the most enriched pathway.In our study, p62 knockdown in U937 cells inhibited cell proliferation and promoted apoptosis , VEGFC, GATA-binding protein 4 , matrix Although AML cells secrete angiogenic factors to remodel the vascular system and gain chemoresistance, anti-angiogenic drugs are generally ineffective in AML treatment. In conclusion, after a detailed examination of miRNA expression in exosomes derived from AML cells, we found that hsa-miR-3064-3p and hsa-miR-339-5p displayed downregulated expression in p62-knockdown cells, compared with control cells. In addition, we demonstrated that several differentially expressed exosomal miRNAs were closely related to multiple GO items and pathways involved in carcinogenesis, indicating that exosomal miRNAs play a key role in AML pathogenesis. This information may aid the development of potential biomarkers for diagnosis and prognosis of AML progression. In the present study, we also found that exosomes derived from AML cells promoted angiogenesis. However, the relationship between exosomal miRNAs and angiogenesis needs to be further investigated. These findings support the notion that promising new treatment strategies may be developed against AML, based on exosomal miRNA analysis.10.7717/peerj.13498/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13498/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.13498/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.13498/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.13498/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.13498/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.13498/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.13498/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.13498/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.13498/supp-10Supplemental Information 10Click here for 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Microbacterium elymi KUDC0405T was isolated from the rhizosphere of Elymus tsukushiensis from the Dokdo Islands. The KUDC0405T strain was Gram-stain-positive, non-spore forming, non-motile, and facultatively anaerobic bacteria. Strain KUDC0405T was a rod-shaped bacterium with size dimensions of 0.3–0.4 × 0.7–0.8 μm. Based on 16S rRNA gene sequences, KUDC0405T was most closely related to Microbacterium bovistercoris NEAU-LLET (97.8%) and Microbacterium pseudoresistens CC-5209T (97.6%). The dDDH  values between KUDC0405T and M. bovistercoris NEAU-LLET and M. pseudoresistens CC-5209T were below 17.3% and 17.5%, respectively. The ANI (average nucleotide identity) values among strains KUDC0405T, M. bovistercoris NEAU-LLET, and M. pseudoresistens CC-5209T were 86.6% and 80.7%, respectively. The AAI (average amino acid identity) values were 64.66% and 64.97%, respectively, between KUDC0405T and its closest related type strains. The genome contained 3,596 CDCs, three rRNAs, 46 tRNAs, and three non-coding RNAs (ncRNAs). The genomic DNA GC content was 70.4%. The polar lipids included diphosphatydilglycerol, glycolipid, phosphatydilglycerol, and unknown phospholipid, and the major fatty acids were anteiso-C17:0 and iso-C16:0. Strain KUDC0405T contained MK-12 as the major menaquinone. Based on genotypic, phylogenetic, and phenotypic properties, strain KUDC0405T should be considered a novel species within the genus Microbacterium, for which we propose the name M. elymi sp. nov., and the type strain as KUDC0405T . Microbacterium was classified by Orla-Jensen S [Microbacterium comprised 157 species, including Microbacterium aerolatum V-73T [M. agarici CC-SBCK-209T [M. album SYSU D8007T [M. algeriense G1T [M. amylolyticum N5T [M. aoyamense KV-492T [M. aquimaris JS54-2T [Microbacterium can be isolated from various sources, such as seawater, desert soil, maize rhizosphere, cow dung, and microfiltered milk. Members of this genus are Gram-stain-positive, rod-shaped, and have an optimum growth temperature of 20–30°C. Here, we report a taxonomic analysis of the novel bacterial strain, KUDC0405T, isolated from the rhizospheric soil of Elymus tsukushiensis, a plant native to the Dokdo Islands . During microbial diversity monitoring in April 2014, rhizospheric soil samples were collected from native plants of the Ulleungdo and Dokdo Islands . E. tsukushiensis var. trasiens (Hack.) Osada is native to the Dokdo Islands and is the dominant plant species on these islands, and its distribution is expanding [Brevibacterium iodinum KUDC1716 [Ochrobactrum lupini KUDC1013 and Novosphingobium pentaromativorans KUDC1065 [The genus Jensen S  based onum V-73T , M. agarU D8007T , M. algeicum N5T , M. aoyaxpanding . DespiteKUDC1716 , Ochroba-4‒10-6) were prepared. A 100 μL aliquot dilution was plated onto R2A and 1/10 diluted tryptic soy agar (TSA)  and incubated at 25°C for 7 days. Morphologically different colonies were selected, and individual colonies were further purified by repeated streaking onto TSA media. The type strains used in this study were obtained from the China General Microbiological Culture Collection Centre (CGMCC) and the Korea Collection for Type Cultures (KCTC). The strains were cultivated on TSA at 25°C and maintained at -70°C in saline solution  supplemented with 15% glycerol (v/v).Plant samples were collected and stored as described previously . The samhttp://macrogen.com/) using the sequencing primers (518F and 800R) and an automated sequencer . The EzBioCloud server (https://ezbiocloud.net/) [The phylogenetics of the isolated strain was determined based on a comparative analysis of the 16S rRNA gene sequence. The 16S rRNA gene sequence was amplified and the PCR products were purified as described previously . UniversRathayibacter rathayi VKM Ac-1601T, which is not affiliated with the genus Microbacterium was used as an outgroup. Trees were rooted and constructed using MEGA-X in the Newick format.All 16S rRNA gene sequences of the closest type strains were aligned using CLUSTAL_W  and the et al. [et al. [T was sequenced using a MinION platform . The reads were assembled de novo using Flye (version 2.9) [T and closely related strains were calculated using the JSpeciesWS website (https://jspecies.ribohost.com/jspeciesws/) [http://enve-omics.ce.gatech.edu/) [http://ggdc.dsmz.de/distcalc2.php) [T and its two closest relatives, M. bovistercoris NEAU-LLET and M. pseudoresistence CC-5209T, using protein sequences annotated by Hyatt et al. [http://automlst.ziemertlab.com) [Chromosomal DNA was extracted in accordance with Sambrook et al.  and was  [et al. . The celion 2.9) . The aution 2.9)  and the ion 2.9)  were useion 2.9) . The aveciesws/) . The avech.edu/) . The dDDlc2.php) . The dDDt et al.  and the t et al. . A multilab.com) .T was observed with a Zentech digital camera  for cell morphology and size, using cells grown on TSA. The cells were treated with 1% osmium tetroxide 25°C for 1 h, and dehydrated with graded series of ethanol , followed by isoamyl acetate. After lyophilization, the samples were coated with platinum , and the cell morphology was observed using a field emission scanning electron microscope .The scanning electron micrograph of strain KUDC0405T and the reference strains (M. bovistercoris NEAU-LLET and M. pseudoresistence CC-5209T) at different temperatures  and different pH values . The pH values were adjusted as described by Kämpfer et al. [Growth capability of strain KUDC0405r et al.  in steriM. bovistercoris NEAU-LLET and M. pseudoresistence CC-5209T, which are related to KUDC0405T, were analyzed under the same conditions. The cell wall peptidoglycan was analyzed using an amino acid analyzer . To analyze the polar lipids, two-dimensional thin layer chromatography (TLC) analysis were used according to Minnikin et al. [T, and reference strains were incubated on TSA at 30°C for 7 days. To determine siderophore production by strain KUDC0405T, chrome azurol S (CAS) media were used as previously described [To determine hydrolysis of starch, urea, Tween 20, 40, 60, and 80, the isolate was cultured on TSA at 30°C for a week, as described by Cowan and Steel . The enzn et al. . The fatescribed , 40.T  was determined as previously described [M. bovistercoris NEAU-LLET, followed by M. pseudoresistence CC-5209T (97.58%), M. resistens NBRC 103078T (97.51%), M. oleivorans NBRC 103075T (97.51%), M. testaceum NBRC 12675T (97.51%), and M. paraoxydans NBRC 103076T (97.30%). A comparison of the preliminary 16S rRNA gene sequences revealed that strain KUDC0405T is related to members of the genus Microbacterium. In the Bayesian inference tree . The complete genome of strain KUDC0405T consisted of a circular chromosome . The genomic DNA G+C content was 70.4%, which is within the range reported for the Microbacterium genus. A total of 3,654 genes were identified, of which 3,018 were protein-coding genes and 52 were RNA genes . T and closely related strains. The genome of strain KUDC0405T displayed 256 subsystems according to genome annotation using RAST. Various metabolic genes were predicted for various metabolic processes, such as the metabolism of amino acids and derivatives (311 genes), carbohydrates (247 genes), cofactors, vitamins, prosthetic groups, pigments (153 genes), proteins (151 genes), nucleosides and nucleotides (106 genes), DNA (60 genes), virulence, disease, and defense (38 genes), membrane transport (33 genes), respiration (33 genes), and other metabolic processes. KUDC0405T did not appear to be motile and the genome contained no genes encoding proteins associated with motility. The OrthoVenn diagram revealed orthologous protein clusters of strain KUDC0405T, M. bovistercoris NEAU-LLET, and M. pseudoresistens CC-5209T  shared 1,510 orthologous protein clusters , T3PKS1 , T3PKS2, RRE-containing , and terpene  . In the case of ANIm, <20% of the genome was aligned for M. bovistercoris NEAU-LLET, and the alignment was assigned as suspicious by the software. In silico, AAI values were analysed at 64.7% and 65.0% in strain KUDC0405T, M. bovistercoris NEAU-LLET, and strain KUDC0405T and M. pseudoresistence CC-5209T, respectively. Also, GGDC results for strains KUDC0405T, M. bovistercoris NEAU-LLET, and M. pseudoresistence CC-5209T were calculated as 17.3% and 17.5% based on formula 2 (identities/HSP length). The ANIb, ANIm, AAI, and dDDH values of strain KUDC0405T compared with those of the closely related strains are presented in T represents a novel species of the Microbacterium genus.The complete genomes determined have been deposited in the NCBI GenBank database under accession number GCF_021582895  . The terT was gram-positive, non-spore forming, non-motile, and grew anaerobically on TSA. Colonies on TSA media were smooth, circular, yellowish-white, and the cells were rod-shaped (0.3–0.4 × 0.7–0.8 μm)  . Growth f plants , 42. Theand urea .T was MK-12. The polar lipids included diphospharidylglycerol, glycolipid, phosphatidylglycerol, an unidentified phospholipid, three unidentified aminolipids, and an unidentified lipid , ornithine (26.8%), alanine (25.4%), and glutamic (15.4%) as cell-wall peptidoglycans. The major fatty acids in KUDC0405T were anteiso-C17:0 (35.2%), iso-C16:0 (16.3%), and iso-C17:0 (8.0%), and the minor components included iso-C15:0 (4.0%), C16:0 (2.6%), anteiso-C15:0 (2.5%), and C18:0 (1.5%). T and the most closely related reference strains. M. bovistercoris NEAU-LLET and M. pseudoresistence CC-5209T presented anteiso-C17:0 and anteiso-C15:0 as major fatty acids. The major fatty acids in the genus Microbacterium were anteiso-C17:0.The predominant menaquinone in KUDC0405ed lipid . DiphospCC-5209T . Strain T represents a novel species of the genus Microbacterium, for which we suggest the name M. elymi sp. nov..To conclude, we suggest that strain KUDC0405T produces siderophores and contains glycine, ornithine, alanine, and glutamic acid as cell-wall peptidoglycans. The polar lipids were diphosphatydilglycerol, glycolipid, phosphatydilglycerol, and phospholipid; the major menaquinone was MK-12; and the major fatty acids were anteiso-C17:0 and iso-C16:0. The genomic DNA GC content was 70.4%.Cells are Gram-stain-positive, catalase- and oxidase- positive, non-spore forming, non-motile, facultatively anaerobic and rod-shaped (0.3–0.4 × 0.7–0.8 μm). Colonies are smooth, circular, yellowish-white, and 3.0–0.4 mm in diameter on TSA with growth for 2 days. Optimal growth occurs at 25–30°C, at pH 7, and 0.5–1.2%NaCl (w/v) on TSA media. Strain KUDC0405T (=KCTC 49411T =CGMCC1.18472T), was isolated from the rhizosphere of E. tsukushiensis collected from the Dokdo Islands, Republic of Korea. The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA gene sequence and genome sequence of KUDC0405T were MT071892 and CP091139, respectively. NCBI accession number for genomes are GCF_021582895.The type strain, KUDC0405de novo assembly are as follows: genome size, 3,610,832 bp; number of contigs, 1; coverage, 119.0 ×.General features of the genome http://jmb.or.kr.Supplementary data for this paper are available on-line only at


Flavobacterium sediminilitoris YSM-43T, isolated from a tidal flat in Yeosu, Republic of Korea. The whole genome consists of one circular chromosome of 3,913,692 bp. A total of 3,599 genes were predicted, comprising 3,537 coding DNA sequences (CDSs), 50 tRNAs, 9 rRNAs, and 3 noncoding RNAs (ncRNAs).Here, we report the complete genome sequence of  Flavobacterium, the type genus of the family Flavobacteriaceae in the phylum Bacteroidetes, is a Gram-negative, yellow-pigmented, and rod-shaped bacterium. Commonly, Flavobacterium thrives in various habitats, including both terrestrial and marine ecosystems (Flavobacterium species have been published (https://lpsn.dsmz.de/genus/flavobacterium)  . Accordicterium) , two Flailitoris , 5. F. sT was cultivated on marine agar 2216 (BD). The cells were collected in a 5-mL Eppendorf tube for DNA extraction. Extraction of genomic DNA was performed using a genomic DNA extraction kit (RBC), following the manufacturer’s instructions. A spin column  was utilized for DNA cleanup. The NanoDrop 2000 UV-visible (UV-vis) spectrometer was used for measuring the ratio of absorbance at 260/280 nm and 260/230 nm. The genomic DNA (gDNA) concentration was measured using a Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Invitrogen) with a Qubit 2.0 fluorometer. The quantity and size distribution of the purified gDNA were calculated using Agilent 2200 TapeStation software (A.01.05)  were assembled de novo using Flye version 2.8.3 with the parameter “asm-coverage 100” (https://github.com/PacificBiosciences/pbbioconda). Then, the genome was rotated using the fixstart method in Circlator version 1.5.5  (YSM-43A.01.05) . For bio N50, 7,54 bp weredata set . As a resee.ca/) . To assisee.ca/) , eggNOG-see.ca/)  (Table 1F. sediminilitoris YSM-43T has been deposited at GenBank under the accession number CP090145. The raw data have been deposited in the SRA under the accession number SRR17867805.The complete genome sequence of


The medical novelty of COVID-19 requires a comprehensive study of its impact on various areas of human health, including mental health.To study the spectrum and severity of psychopathological disorders in previously healthy patients of different age groups who have had moderate and severe COVID-19 pneumonia.Immediately after stabilization of the physical condition, patients completed the Symptom Checklist-90-R, designed to assess 11 parameters: somatization (SOM), obsessive-compulsive (OS), interpersonal sensitivity (INT), depression (DEP), anxiety (ANX), hostility (HOS), phobic anxiety (PHOB), paranoid ideas (PAR), psychoticism (PSY). Patients with cognitive impairment were excluded.The study involved 148 patients aged from 26 to 84 years. In the general sample, psychopathological symptoms were detected mainly on the SOM, DEP, ANX, HOS scales. To a lesser extent - on the INT and PAR scales; were practically not determined on the PSY and PHOB scales. Most of the symptoms are significantly more intense in patients over 46 years old (n = 129) compared with the younger population . Older patients according to SOM revealed 1.23 points (IQR 0.5) versus 0.85 (IQR 0.7) among young people, DEP - 0.88 (IQR 0.44) vs. 0.47 (IQR 0.44), ANX - 0.66 (IQR 0.44) vs. 0.43 (IQR 0.29), OS - 0.55 (IQR 0.5) vs. 0.31 (IQR 0.25) and HOS - 0.46 (IQR 0.34) vs. 0.29 (IQR 0.09).Patients recovering from severe COVID-19 pneumonia require psychiatric evaluation and subsequent differentiated psychotherapeutic rehabilitation, especially for the age group over 46.No significant relationships.