Μg Of Total RNA Research Articles

First Report of Sugarcane Mosaic Virus Infecting Goose Grass in Shandong Province, China.

Sugarcane mosaic virus (SCMV genus Potyvirus, family Potyviridae) can infect maize, sugarcane, sorghum, other graminaceous crops, and some weed species (Alegria et al., 2003; Achon et al., 2007). In August 2023, the leaves of goose grass (Eleusine indica) plants surrounding maize fields in a village of Liaocheng City, Shandong Province, China showed mosaic and chlorotic symptoms (26%, 11 of 43 grasses; Figure S1). Three symptomatic goose grass samples were selected and pooled for total RNA isolation using TRIzol reagent (Tiangen, Beijing, China). A small RNA library was created using 2.0 μg of total RNA and the mirVana miRNA Isolation Kit, followed by size selection (18-28 nt), adapter ligation, purification, reverse transcription (RT), and polymerase chain reaction (PCR) enrichment. High-throughput sequencing (HTS) was then performed on a HiSeq 2500 platform (Illumina, San Diego, CA, USA). The adapter sequences were removed and the reads were assembled de novo into larger contigs using ABySS software v. 1.9.0 with a k-mer of 32. Fifty-one contigs were obtained after the reads were spliced and screened (alignment length > 30 bp; e-value ≤ 0.05). The contigs were compared with viral sequences in GenBank using local BLASTn. Thirty-four contigs (34-64 nt) had the highest identities (97.18-100%) with the SCMV genome sequence, covering approximately 12.8% of the SCMV genome (Table S1). The low coverage of small contigs mapping to the SCMV genome in the HTS results may be attributed to variations in sequencing depth and sample preparation quality, biological aspects of the virus affecting siRNA production and detection, as well as the variability in viral genome and its size (Golyaev et al., 2019; Valenzuela et al., 2022). The other 17 contigs did not align to any plant virus sequences, but aligned to plant sequences, including Phragmites australis and Panicum virgatum. Potyvirus-degenerated primers PotyF (5'-ATGGTHTGGTGYATHGARAAYGG-3') and PotyR (5'-TGCTGCKGCYTTCATYTG-3') (Marie-Jeanne et al. 2000) were used in RT-PCR to detect SCMV in symptomatic leaves, yielding a ~300 bp amplicon. Sanger sequencing and BLASTn analysis confirmed the 97.98% nucleotide identity with SCMV isolate BJ (GenBank accession No. AY042184.1). The sequence was deposited in GenBank under accession number OR777055. In addition, specific SCMV primers SCMV-F (5'- TCCGGAACTGTGGATGCA-3') and SCMV-R (5'- GTGGTGCTGCTGCACTCCC-3') (coat protein region, 939 bp) detected the virus in all 11 symptomatic goose grass leaves, with no detection in asymptomatic leaves. Inoculation tests using extracts from symptomatic goose grass on maize plants resulted in mosaic symptoms (7 of 15 plants) at 4-6 days post-inoculation (Figure S2 and 3). However, no symptoms were observed in maize plants following inoculation with leaf extracts from healthy goose grass. RT-PCR confirmed the presence of SCMV in the diseased maize plants. Sequencing analysis revealed that all amplified fragments shared 100% identity with the partial CP-encoding sequence of SCMV. Taken together these results support the presence of SCMV in symptomatic goose grass. To the best of our knowledge, this is the first report of SCMV in E. indica in China. In general, potyviruses can be easily transmitted in multiple ways including aphid vectors, grafting, and wounding. Therefore, investigating SCMV in goose grass is crucial for developing integrated strategies to prevent its transmission to economically important plants such as maize.

Study of head and neck squamous cell carcinoma transcriptome after proton therapy

Aim. To evaluate changes in the transcriptome of head and neck squamous cell carcinoma (HNSCC) tissue cells in patients after proton therapy.Materials and methods. Biopsy material obtained from 3 HNSCC patients before and after proton therapy at a total dose of 10 isoGy was homogenized, purified, and concentrated. Then total RNA was isolated with further purification and concentration with the RNA Clean & Concentrator kit (Zymo Research). Library quantitation was assessed using the Qubit 2.0 instrument (Invitrogen, Life Technologies). After isolation of 1 μg total RNA for sequencing, libraries were prepared on the Illumina platform using the TruSeq RNA Sample Prep Kit v2 with a 10-cycle enrichment step according to the manufacturer’s recommendations. The quality of RNA and the resulting libraries was checked using the Agilent 2100 Bioanalyzer system (Agilent Tec. Inc., USA). The RIN parameter for RNA was at least 7. The library concentration was assessed by real-time PCR on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). Final libraries were pooled in equimolar ratios before sequencing on the Illumina HiSeq 2500 platform using 50 base-pair paired-end reads. The Q20 parameter for all samples was > 97%, and the number of reads averaged 60.2 million per sample. Raw reads were processed using the RTA 1.17.21.3 and Casava 1.8.2 (Illumina). The enrichment analysis was performed using the PANTHER 17.0 software.Results. The transcriptome analysis of HNSCC after proton radiation therapy (5 x 2 isoGy) at a total dose of 10 isoGy revealed 1,414 significantly differentially expressed genes. The 10 most and least expressed genes and their associated signaling pathways were identified. A number of signaling pathways associated with the underexpressed genes were detected in HNSCC after proton therapy, such as: STAT5; PD-1 signaling pathway; marked MET-mediated activation of PTK2 signaling pathway, PDGF signaling; CD22-mediated regulation of BCR; and FCERI-mediated MAPK activation. In addition to the above signaling pathways, activation of collagen degradation, FCGR3A-mediated phagocytosis, and FCGR3A-mediated interleukin (IL)-10 synthesis are of interest. In the enrichment analysis among highly expressed genes, keratinization and biological oxidation processes were activated in HNSCC tissues after proton therapy.Conclusion. Proton therapy in HNSCC leads to overexpression of genes involved in the regulation of keratinization and biological oxidation processes as well as to underexpression of genes associated with suppression of signaling pathways: STAT5, PD-1, MET-mediated activation of PTK2 signaling pathway, PDGF signaling; CD22-mediated regulation of BCR; FCERI-mediated MAPK activation, collagen degradation, FCGR3A-mediated phagocytosis activation, and FCGR3A-mediated IL-10 synthesis. All signaling pathways of underexpressed genes function in HNSCC cells if there is no negative influence on the tumor from outside (irradiation or delivery of antitumor drugs). The predominance of suppressed signaling pathways over activated ones most likely indicates a decrease in the functional potential of cells after proton therapy. The dose-dependence of PT effects necessitates further study of changes in cellular and molecular-genetic signatures of HNSCC after proton irradiation with different doses.

A low-cost, low-input method establishment for m6A MeRIP-seq.

N6-methyladenosine (m6A) is a highly prevalent modification found in mammal mRNA molecules that plays a crucial role in the regulation of cellular function. m6A RNA immunoprecipitation sequencing (MeRIP-seq) has been frequently used in transcriptomics research to identify the location of m6A. MABE572 (Millipore) is the most widely utilized and efficient anti-m6A antibody for MeRIP-seq. However, due to the high dose and price of this antibody, which has also been taken off the market, we discovered that CST's anti-m6A antibody can be used instead of MABE572 to map the m6A transcriptome. In the present study, we performed different concentrations of the CST anti-m6A antibodies with the corresponding initiation RNA of HEK293T cells, 2.5 μg antibody with 1 μg total RNA, 1.25 μg antibody with 0.5 μg total RNA, and 1.25 μg antibody with 0.1 μg total RNA. By comparing the m6A peak calling, enriched motifs, alternative splicing events, and nuclear transcripts modified by m6A between the CST and Millipore libraries, it was found that the CST library presented similar data to Millipore, even at incredibly low doses. The volume and cost of antibodies are significantly reduced by this refined MeRIP-seq using CST antibody, making it convenient to map future large-scale sample m6A methylation.

First report of Nerine yellow stripe virus infecting crinium lily (Crinium sp.) in Texas.

Crinum sp. (family Amaryllidaceae) is an ornamental flower bulb that is commonly called crinum lily, cape lily, cemetery plant, spider lily, and swamp lily. In April 2023, two plants of Crinum sp. var. Maiden's Blush with yellow stripe symptoms (Fig. S1) were submitted to the Texas Plant Virus Diagnostic Laboratory, Weslaco, TX for virus diagnosis. Due to the resemblance of the observed symptoms to those described for potyviruses infecting ornamental flower bulbs (Pearson et al. 2009), total RNA extracts were made from each sample using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 µg total RNA per sample with Oligo(dT) primers using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, USA) as recommended by the manufacturer. A 2µL aliquot of each cDNA template was initially subjected to PCR using the generic primer pair CIFor/CIRev (Ha et al., 2008) that targets a fragment of the cylindrical inclusion (CI) body of potyviruses. The expected ~700 bp DNA band was amplified from both samples using the Taq DNA polymerase, dNTPack kit (Sigma-Aldrich). The amplicons were cloned and sequenced (three recombinant clones per sample) as described by Hernandez et al. (2021) and the BLASTX analyses of the consensus sequence (GenBank acc. no. OR137018) returned significant hits only to nerine yellow stripe virus (NeYSV; Potyvirus, Potyviridae) at 100% query coverage. To further confirm the results, another pair of universal primers (Jordan et al. 2011) was used to amplify the expected ∼1,600 bp product specific to the partial nuclear inclusion body (NIb), coat protein (CP) cistron, and 3' untranslated region of potyviruses from the same samples. The amplicons were similarly cloned, and a consensus sequence obtained (OR137019). In pairwise comparisons, the partial CI sequence of NeYSV from Texas (NeYSV-TX; OR137018) shared 83% nucleotide (nt)/93% amino acids (aa) identities with the corresponding sequences of NeYSV isolate 63 (MT396083) from the United Kingdom. The partial (649 nt) NIb sequences of NeYSV-TX (OR137019) and the complete CP (OR137019) of NeYSV-TX shared 77-94%/88-94% and 83-99%/89-98% nt/aa identities with the corresponding sequences of global NeYSV isolates that were retrieved from GenBank. Phylogenetic analysis revealed a closer relationship between NeYSV-TX and the isolates Stenomesson (EU042758) and DC (MG012805) from the Netherlands and USA, respectively based on the partial NIb and CP cistrons (Fig. S2), suggesting that NeYSV-TX may have been introduced from foreign and/or domestic sources. NeYSV has been documented previously from the United Kingdom, the Netherlands, Australia, New Zealand, and India; its first report from the United States was a decade ago from Amaryllis belladonna in California (Guaragna et al. 2013). To the best of our knowledge, this is the first report of NeYSV in Texas, thus expanding the geographical range of the virus in the USA. Anecdotal information from the sample submitter implicated infected crinum lily bulbs as the likely source of NeYSV introduction into the property, with subsequent vegetative propagation of plants resulting in 100% incidence of symptomatic lilies (n>100) over time. Thus, the results underscore the importance of ensuring that only virus-free vegetative plant materials are distributed and propagated by florists to curtail virus spread.

Culture of leukocyte-derived cells from human peripheral blood: Increased expression of pluripotent genes OCT4, NANOG, SOX2, self-renewal gene TERT and plasticity.

There are few stem cells in human peripheral blood (PB). Increasing the population and plasticity of stem cells in PB and applying it to regenerative medicine require suitable culture methods. In this study, leukocyte populations 250 mL of PB were collected using a blood separator before that were cultured in optimal cell culture medium for 4 to 7 days. After culturing, stemness characteristics were analyzed, and red blood cells were removed from the cultured cells. In our results, stemness markers of the leukocyte populations Sca-1+ CD45+, CD117+ CD45+, and very small embryonic-like stem cells CD34+ Lin- CD45- and CXCR4+ Lin- CD45- were significantly increased. Furthermore, the expression of stem cell genes OCT4 (POU5F1), NANOG, SOX2, and the self-renewal gene TERT was analyzed by quantitative real-time polymerase chain reaction in these cells, and it showed a significant increase. These cells could be candidates for multi-potential cells and were further induced using trans-differentiation culture methods. These cells showed multiple differentiation potentials for osteocytes, nerve cells, cardiomyocytes, and hepatocytes. These results indicate that appropriate culture methods can be applied to increase expression of pluripotent genes and plasticity. Leukocytes of human PB can be induced to trans-differentiate into pluripotent potential cells, which will be an important breakthrough in regenerative medicine.

Micrornas Analysis in Patients with Myelodysplastic Syndrome. Possible Implications in Risk Stratification

Background. MicroRNAs (miRNAs) are significant regulators of human hematopoietic stem cells (HSC), and their deregulation contributes to hematological malignancies. Systematic analysis of miRNA expression and function during hematopoiesis has revealed regulatory loops where miRNAs contribute to hematopoietic differentiation and function. Among the identified miRNAs, miR-155 can regulate myelopoiesis and erythropoiesis and its overexpression along with the overexpression of miR-125 and miR-181 has been suggested to have functional effects in the regulation of HSC homeostasis. Myelodysplastic syndromes (MDS) are characterized by dysfunctional HSC, ineffective blood cell production, progressive marrow failure, and exhibit a significant risk of progression to acute myeloid leukemia (AML). Although the pathogenesis of MDS has not been fully understood, various alterations of microRNAs (miRNAs) have been reported in MDS. There is recent evidence that the miRNAs mediated post-transcriptional control of gene expression is key component in the pathogenesis of MDS and AML. Several studies have reported that differentially expressed microRNAs are associated with the transformation of MDS to AML and clinical outcomes. Aim. In this study we examined the potential prognostic impact of six previously described miRNAS (miR-181a-2-3p, miR-124-3p, miR-550a-3p, miR-155-5p, miR-151a-3p, and miR-125b-5p) on the pathogenesis of MDS. Methods. Total RNA was extracted from diagnostic bone marrow samples of 41 MDS patients. Patients were stratified per the revised International Prognostic Scoring System (IPSS-R). After polyadenylation of 1 μg total RNA by poly(A) polymerase and subsequent reverse transcription with an oligo-dT adapter primer, miRNAs transcript levels were determined using a reverse transcription quantitative real-time PCR method, based on SYBR Green chemistry. U6 was used as reference gene. Relative microRNA transcript levels were calculated by the 2-ΔΔct method. The IBM SPSS statistics, version 26 (IBM Corporation, North Castle, NY, USA) was used for the statistical analysis of the results. Results. The basic characteristics of the patients are shown in Table 1. Of the microRNAs analyzed in this study, miR-125b-5p, miR-155-5p, miR-181a-2-3p transcript levels were significantly elevated in higher (intermediate, high, and very high) risk MDS than in lower (very low and low) risk MDS (p=0.012, p=0.010, and p=0.024 respectively). MiR-125b-5p, miR-151a-3p, miR-155-5p, miR-181a-2-3p, and miR-550a-3p presented a positive correlation with the percentage of bone marrow blasts [p(0.001-0.05)], while a negative correlation was observed between the expression levels of mir-151-a-3p, miR181a-2-3p, miR-550a-3p, and the neutrophil count (p=0.05, 0.013, 0.010, and 0.007 respectively). None of the remaining factors that were tested (MDS type per the 2016 WHO classification, gender, age at diagnosis, hemoglobin levels, platelet count, and cytopenias) were correlated with the miRNAs levels. Discussion. In this study we demonstrated the notable difference in the pattern of miRNA expression between lower and higher risk MDS patients. It has been shown that deregulation of miR-155-5p and miR-181a-2-3p may promote aberrant hematopoietic stem cell proliferation, differentiation, and progression to AML. Moreover, mice transplanted with miR-155-5p transfected stem cells developed a myeloproliferative disorder with abnormal granulocyte morphology analogous to MDS, suggesting a role in the higher risk MDS phenotype. Finally, it has been reported that miR-181a-3p was strongly expressed in the cytoplasm of high risk MDS cells, in contrast to low risk samples which displayed reduced levels. The identification of miRNAs that distinguish lower risk from higher risk MDS indicates that microRNA expression profile may be exploited to assist with making treatment decisions. Given that certain miRNAs can discriminate MDS subtypes and risk groups, it is not unlikely to think that an miRNA signature could be a useful tool for MDS risk stratification. Based on our current findings and other previously reported results, we continue the study in a larger cohort of patients. Targeting miRNAs may provide new treatment options for MDS Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Role of miR-21-5p/FilGAP axis in estradiol alleviating the progression of monocrotaline-induced pulmonary hypertension.

BackgroundAberrant expression of microRNAs (miRNAs) has been associated with the pathogenesis of pulmonary hypertension (PH). It is, however, not clear whether miRNAs are involved in estrogen rescue of PH.MethodsFresh plasma samples were prepared from 12 idiopathic pulmonary arterial hypertension (IPAH) patients and 12 healthy controls undergoing right heart catheterization in Shanghai Pulmonary Hospital. From each sample, 5 μg of total RNA was tagged and hybridized on microRNA microarray chips. Monocrotaline‐induced PH (MCT‐PH) male rats were treated with 17β‐estradiol (E2) or vehicle. Subgroups were cotreated with estrogen receptor (ER) antagonist or with antagonist of miRNA.ResultsMany circulating miRNAs, including miR‐21‐5p and miR‐574‐5p, were markedly expressed in patients and of interest in predicting mean pulmonary arterial pressure elevation in patients. The expression of miR‐21‐5p in the lungs was significantly upregulated in MCT‐PH rats compared with the controls. However, miR‐574‐5p showed no difference in the lungs of MCT‐PH rats and controls. miR‐21‐5p was selected for further analysis in rats as E2 strongly regulated it. E2 decreased miR‐21‐5p expression in the lungs of MCT‐PH rats by ERβ. E2 reversed miR‐21‐5p target gene FilGAP downregulation in the lungs of MCT‐PH rats. The abnormal expression of RhoA, ROCK2, Rac1 and c‐Jun in the lungs of MCT‐PH rats was inhibited by E2 and miR‐21‐5p antagonist.ConclusionsmiR‐21‐5p level was remarkably associated with PH severity in patients. Moreover, the miR‐21‐5p/FilGAP signaling pathway modulated the protective effect of E2 on MCT‐PH through ERβ.

Abstract 523: Lipid-associated Plasminogen Is A Ribonucleoprotein Carrier Of Extracellular Small RNAs In Atherosclerosis

Small RNAs (sRNA) are exchanged between cells and tissues by various lipid and protein carriers. Extracellular sRNAs regulate gene expression in recipient cells, including activation of pattern recognition receptors and inflammatory signaling pathways. Based on size-exclusion chromatography (SEC) fractionation of human plasma, a large proportion of extracellular sRNAs are detected in small lipid-associated ribonucleoproteins (50-150 kDa), a class of carriers that have been largely overlooked. Sequential fractionation approaches, mass spectrometry, enzymatic activity assays, and western blotting all support that lipid-associated human plasminogen (PLG) co-purifies with sRNAs in plasma. PLG transports approximately 10 μg total RNA/ mg protein in plasma. Depletion of PLG from plasma by lysine-sepharose resin was also found to reduce plasma miRNA levels. Based on microscale thermophoresis assays, PLG was determined to have moderate binding to single-stranded sRNAs (K d =1.8μM). Immune cells may be a source of sRNAs, as macrophages were found to export both host and non-host sRNAs to PLG in vitro . To identify PLG-associated sRNAs, sRNA sequencing was completed, and results showed that PLG host sRNA are predominantly derived from parent rRNAs (rDR), tRNAs (tDR), yRNA (yDRs), and microRNAs (miRNA). Furthermore, we found that host sRNAs on PLG are significantly altered in subjects with familial hypercholesterolemia (hetFH), including 22 miRNAs, 5 tDRs, and 10 yDRs. PLG was also found to transport many non-host microbial sRNAs derived from bacteria, fungi, and viruses in the microbiome and environment; and genome counts for many of these microbial species were significantly altered on PLG isolated from hetFH subjects. PLG was found to cause dramatic gene (mRNA) expression changes in treated-macrophages; however, it is unknown what fraction of these changes are associated with PLG-sRNA delivery. Collectively, results suggest that PLG transports high levels of extracellular sRNAs from host and non-host species that are sensitive to hypercholesterolemia and disease associated changes. Although the impact of PLG-sRNAs remains to be determined, they hold great potential as therapeutic targets for inflammation.

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Epigenetic modifications of tumor necrosis factor-alpha in joint cartilage tissue from osteoarthritis patients - CONSORT.

Background:Osteoarthritis (OA) remains one of the most common osteopathy for centuries, which can be attributed to multiple risk factors including mechanical and biochemical ones. More and more studies verified that inflammatory cytokines play important roles in the progression of OA, such as tumor necrosis factor-alpha (TNF-α). In this study, we aimed to investigate the relationship between epigenetic manifestations of TNF-? and the pathogenesis of OA.Methods:Totally, 37 OA patients’ cartilage was collected through the knee joint and 13 samples of articular cartilage as healthy control was collected through traumatic amputation. Real-time PCR, Western blot and ELISA analysis were performed to observe the expression of target genes and proteins in collected samples.Results:Compared with the healthy control group, TNF-? was over-expressing in cartilage which was collected from OA patients. DNA hypomethylation, histone hyperacetylation and histone methylation were observed in the TNF-? promoter in OA compared with normal patients, and we also studied series of enzymes associated with epigenetics. The results showed that by increasing DNA methylation and decreasing histone acetylation in the TNF-? promoter, and TNF-? over-expression in OA cartilage was suppressed, histone methylation has no significant correlation with OA.Conclusion:In conclusion, the changes of epigenetic status regulate TNF-α expression in the cells, which are pivotal to the OA disease process. These results may give us a better understanding of OA and may provide new therapeutic options.

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

AbstractAn athermal approach to mRNA enrichment from total RNA using a self‐immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six‐atom spacing between bases which allowed TENA to selectively base‐pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL−1. Self‐immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT‐qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT‐qPCR and other downstream molecular biology applications.

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids.

An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL-1 . Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.

A novel regulatory role of trappc9 and L-plastin in osteoarthritis

A novel regulatory role of trappc9 and L-plastin in osteoarthritis

Immobilized Ag-nanoparticles (iNPs) for environmental applications: Elucidation of immobilized silver-induced inhibition mechanism of Escherichia coli

Although silver nanoparticles (AgNPs) appear to be promising for certain medical/pharmaceutical applications, they present significant disadvantages when it comes to environmental applications due to the need for recovery of the metal (Ag) which is considered hazardous for the environment. The present study examines the antimicrobial properties and mechanism of action of immobilized (on Al2O3) silver nanoparticles’ (1 wt% Ag-iNPs) regarding the treatment of E. coli microbial solutions. Antimicrobial experiments were conducted in semi-batch mode using a three-phase continuous flow stirred tank reactor. Every treated sample was taken from the outlet of the reactor for the time intervals of 0, 5 and 25 min. To ensure that the bactericidal property of Ag-iNPs is not attributed to the dissolution of surface silver (as free Ag+), a suitable Ag+ scavenger was used as already described in our earlier studies. Regarding the molecular analysis performed under this study, the regulation of key enzymes involved in bactericidal activity of E. coli, was examined, after their treatment either with immobilized silver nanoparticles (Ag-iNPs) or AgNO3. Specifically, 50 mL sample for each case was centrifuged (10 min at 10,000 rpm) and the pellet (≈109 cells) was immediately subjected to total RNA extraction. For real-time RT-qPCR analyses, 1 μg of total RNA was converted into cDNA. It was found that PldA gene, which encodes outer membrane’s phospholipase A (OMPLA), was up-regulated after 5 and 25 min of treatment with Ag-iNPs. OMPLA’s activation appears to be the initial step of Ag-iNPs bactericidal mechanism that ultimately leads to the creation of holes on the outer membrane (OM), irreversibly disturbing the cells’ respiration cycle. In addition, after treatment with Ag-iNPs, Blue copper oxidase CueO (encoded by cueO gene) was found to be over-produced and appears to play a key-role in the oxidative transfer of silver from the surface of Ag-iNPs to the cell. This leads to the Ag-induced displacement of copper from its native protein sites such as CusS/CusR, a fact that causes the release of labile copper and ROS inside the cell. In addition, TEM analysis of bacteria treated with Ag-iNPs revealed severe morphological changes (“holes”) on bacterial outer membrane, indicating that there is an intermediate step in the proposed antimicrobial mechanism which takes place on the surface of Ag-iNPs.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Small RNA Isolation and Library Construction for Expression Profiling of Small RNAs from Neurospora crassa and Fusarium oxysporum and Analysis of Small RNAs in Fusarium oxysporum-Infected Plant Root Tissue.

Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of sRNAs is necessary. We developed a protocol for isolation and library construction of sRNAs of 20-30nt for deep sequencing in two filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300μg total RNA, sRNA was isolated by size-fractionation and ligated with adapters and amplified by RT-PCR for deep sequencing. Sequence analysis of several cDNA clones showed that the cloned sRNAs were not tRNAs and rRNAs and were fungal genome-specific. In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.

Lesional Inflammatory Profile in Hidradenitis Suppurativa Is Not Solely Driven by IL-1

Lesional Inflammatory Profile in Hidradenitis Suppurativa Is Not Solely Driven by IL-1

First Report of Tobacco Vein Banding Mosaic Virus Naturally Infecting Black Nightshade (Solanum nigrum) in China

Black nightshade (Solanum nigrum L. var. humile [Bernh.]) is a common solanaceous plant that is an arable weed and also used in herbal medicine in China (Wang et al. 2017b; Zhao et al. 2018). The weed occurs commonly in a wide variety of habitats, including orchards, fields, villages, and roadsides in southwest provinces in China. In May 2019, about 3% of black nightshade plants growing in a tomato field at Kunming city in Yunnan province in China were found showing typical symptoms of viral disease (chlorosis, mosaic, leaf twisting, and plant stunting). To identify the suspected virus, one plant with severe symptoms was chosen for small RNA sequencing. Total RNA was extracted from leaves of symptomatic tissues by TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA), and a small RNA library was constructed and then sequenced by high-throughput sequencing (HTS) on an Illumina HiSeq 2500 platform. The analysis generated approximately 11.37 million Illumina reads (15 to 40 nt in length), of which more than 0.082 million reads were mapped to the tobacco vein banding mosaic virus (TVBMV) genome (reference sequence accession no. NC_009994.1). No other viruses and viroids were detected. Further observation by transmission electron microscopy showed that the negatively stained crude sap of two symptomatic plants contained flexuous, filamentous virus particles about 600 to 900 nm in length, typical of a potyvirus. To further confirm the results, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA), first-strand cDNA was synthesized from 1 µg of total RNA using an oligo dT primer and a random primer mix, and then fragments of the TVBMV-CP gene were amplified with specific primers (F, 5′-AATGACGAACAGACAGTTGATGCTG-3′; R, 5′-CACGCCACTCACACCAAGTAGG-3′; designed from the CP gene of an HTS-generated clone) by 2×T5 Super PCR Mix (Tsingke BioTech, China). After cloning into a pGEM-T vector (Promega Biotech, U.S.A.), six clones of each fragment were randomly chosen for Sanger sequencing (Tsingke BioTech). The clone sequences were identical, and this partial TVBMV sequence was then submitted to GenBank (accession no. MN331841). This new sequence was most similar to Chinese isolate XDBB-01 (95.1% nucleotide identity to the CP mRNA, accession MG880266.1, and 98.2% amino acid identity to the corresponding coat protein AYG97953.1). Ten additional black nightshade plants with similar symptoms and another three asymptomatic plants were randomly selected and tested for the presence of TVBMV using the same primers. All 10 symptomatic plants tested positive for TVBMV, whereas the asymptomatic plants tested negative by reverse transcription PCR (RT-PCR) (data not shown). Transmissibility of TVBMV from black nightshade to tomato (cultivar KP13) by aphids (Myzus persicae), which were present widely in the tomato fields in the region, was tested according to the method reported by Yu et al. (2007). At 21 days postinoculation, 10 out of 20 inoculated plants showed leaf twisting symptoms. RT-PCR confirmed the presence of TVBMV in these plants but not in plants without symptoms. Results from three biological repeats were consistent, demonstrating that black nightshade can serve as an effective inoculum source for TVBMV. TVBMV has been reported to infect tobacco, potato, Datura stramonium, wild eggplant, tomato, and sesame in China (Wang et al. 2017a). To our knowledge, this is the first report of TVBMV infecting S. nigrum in natural field conditions. Our findings indicate that S. nigrum in the field could be a reservoir of TVBMV to infect tomato and cause viral disease in tomato fields.

The Validation of the BCR/ABL1-like Predictor across Laboratories Shows Reproducibility of Results

The Validation of the BCR/ABL1-like Predictor across Laboratories Shows Reproducibility of Results