Preliminary Sequence Research Articles

The AP2 Transcription Factor BrSHINE3 Regulates Wax Accumulation in Nonheading Chinese Cabbage.

Wax is an acellular structural substance attached to the surface of plant tissues. It forms a protective barrier on the epidermis of plants and plays an important role in resisting abiotic and biotic stresses. In this paper, nonheading Chinese cabbage varieties with and without wax powder were observed using scanning electron microscopy, and the surface of waxy plants was covered with a layer of densely arranged waxy crystals, thus differentiating them from the surface of waxless plants. A genetic analysis showed that wax powder formation in nonheading Chinese cabbage was controlled by a pair of dominant genes. A preliminary bulked segregant analysis sequencing (BSA-seq) assay showed that one gene was located at the end of chromosome A09. Within this interval, we identified BraA09000626, encoding an AP2 transcription factor homologous to Arabidopsis AtSHINE3, and we named it BrSHINE3. By comparing the CDS of the gene in the two parental plants, a 35 bp deletion in the BrSHINE3 gene of waxless plants resulted in a frameshift mutation. Tissue analysis showed that BrSHINE3 was expressed at significantly higher levels in waxy plant rosette stage petioles and bolting stage stems than in the tissues of waxless plants. We speculate that this deletion in BrSHINE3 bases in the waxless material may inhibit wax synthesis. The overexpression of BrSHINE3 in Arabidopsis induced the accumulation of wax on the stem surface, indicating that BrSHINE3 is a key gene that regulates the formation of wax powder in nonheading Chinese cabbage. The analysis of the subcellular localization showed that BrSHINE3 is mainly located in the nucleus and chloroplast of tobacco leaves, suggesting that the gene may function as a transcription factor. Subsequent transcriptome analysis of the homology of BrSHINE3 downstream genes in nonheading Chinese cabbage showed that these genes were downregulated in waxless materials. These findings provide a basis for a better understanding of the nonheading Chinese cabbage epidermal wax synthesis pathway and provide important information for the molecular-assisted breeding of nonheading Chinese cabbage.

Upregulation of lncRNA HITT promotes cell apoptosis by suppressing the maturation of miR-602 in gastric cancer.

It has been reported that HITT can inhibit colon cancer. However, the role of HITT in gastric cancer (GC) is unknown. Our preliminary sequencing data revealed the altered expression of HITT in GC and its close correlation with miR-602, suggesting the involvement of HITT and its potential interaction with miR-602 in GC. This study explored the role of HITT and its crosstalk with miR-602 in GC. In this study, the expression of HITT, premature and mature miR-602 in paired GC and normal tissues (62 patients) was detected by RT-qPCR. RNA pull-down assay was performed to evaluate the direct interaction between HITT and mature miR-602. The subcellular location of HITT was assessed by nuclear fractionation assay. The role of HITT in regulating miR-602 maturation was explored by overexpression assay. Cell apoptosis was analyzed by flow cytometry. Our data illustrated that HITT was highly upregulated and mature miR-602 was downregulated in GC. No alteration in premature miR-602 in GC was observed. HITT was located in both nucleus and cytoplasm, and it can directly interact with miR-602. In addition, overexpression of HITT in GC cells increased the expression levels of mature miR-602 but not premature miR-602. Overexpression of HITT further increased GC cell apoptosis and suppressed the role of miR-602 in inhibiting GC cell apoptosis. In conclusion, HITT may promote GC cell apoptosis by suppressing the maturation of miR-602.

Arid2-IR downregulates miR-132-3p through methylation to promote LPS-induced ALI in pneumonia

Objective Arid2-IR is a long non-coding RNA (lncRNA) that promotes renal injury, while its role in lipopolysaccharides (LPS)-induced acute lung injury (ALI) is unknown. Our preliminary sequencing analysis revealed an inverse correlation of Arid2-IR and miR-132-3p, which is known to suppress LPS-induced ALI. Therefore, Arid2-IR and miR-132-3p may interact with each other to participate in LPS-induced ALI in pneumonia. This study aimed to investigate the interaction between Arid2-IR and miR-132-3p in ALI induced by pneumonia. Materials and methods Plasma samples were obtained from patients with pneumonia (n = 98) and healthy controls (n = 98) to detect the expression of circulating Arid2-IR and miR-132-3p. The correlation between them was analyzed using Pearson’s correlation coefficient. The crosstalk between them in human bronchial epithelial cells (HBEpC) was analyzed through overexpression assay. MSP was applied to determine the methylation of the miR-132-3p gene. Cell viability was evaluated by 2,5-diphenyl-2H-tetrazolium bromide assay. Results Arid2-IR was highly upregulated in pneumonia group, while the expression levels of miR-132-3p decreased in pneumonia group compared to that in the controls. Arid2-IR and miR-132-3p were inversely correlated across patient samples. Overexpression of Arid2-IR decreased the expression levels of miR-132-3p in HBEpCs and increased the methylation of miR-132-3p gene. Arid2-IR suppressed the role of miR-132-3p in increasing the viability of HBEpCs induced by LPS. Discussion and conclusion Arid2-IR is upregulated in pneumonia and may downregulate miR-132-3p by increasing its methylation to decrease cell viability, thereby promoting LPS-induced ALI in pneumonia.

Diabetes regulated anti-inflammatory lncRNA is overexpressed in triple-negative breast cancer and predicts chemoresistance and tumor recurrence

ABSTRACT Diabetes regulated anti-inflammatory lncRNA (DRAIR) has been characterized as a critical player in type 2 diabetes mellitus. However, its role in other human diseases is unclear. Our preliminary sequencing analysis revealed an altered DRAIR level in triple-negative breast cancer (TNBC). We then explored the involvement of DRAIR in TNBC. In this study, plasma was collected from healthy controls (n = 60) and TNBC patients (n = 60). Non-tumor tissues and paired TNBC were also collected from TNBC patients. All TNBC patients received surgical resection of the primary tumors and chemotherapy. The 60 TNBC patients were divided into high and low plasma DRAIR level groups with median plasma DRAIR level as the cutoff value, and the occurrence of chemoresistance after treatment and tumor recurrence during a 5-year follow-up was monitored. Cell proliferation and viability were analyzed with BrdU assay and MTT assay, respectively. We found that plasma DRAIR levels were higher in TNBC patients than in controls. In addition, DRAIR expression in TNBC tissues was also significantly increased in TNBC tissues compared to the paired non-tumor tissues. Plasma DRAIR was positively and significantly related to DRAIR levels in TNBC tissues but not in non-tumor tissues. Patients in the high plasma DRAIR level group showed a significantly higher rate of chemoresistance after treatment and tumor recurrence during the follow-up. DRAIR promoted tumor cell proliferation and increased cell viability under doxorubicin treatment. Therefore, DRAIR is overexpressed in TNBC and predicts chemoresistance and tumor recurrence.

EAE suppression caused by Δ-8-THC treatment is due to downregulation of CNS infiltrating CD4+ T-cells, proinflammatory cytokines and apoptotic genes expression

Abstract Δ-8-THC a naturally occurring cannabinoid found in trace amounts in Cannabis plants. It possesses potent anti-inflammatory properties along with several other medical benefits. Δ-8-THC has been shown to induce immune system homeostasis particularly in individuals with autoimmune disorders by reducing the activity of T cells and B cells. In current study, we used the experimental model of MS to better understand how Δ-8-THC suppresses neuroinflammation in immune cell-mediated disease called experimental autoimmune encephalomyelitis (EAE). Δ-8-THC administration reduced significantly the clinical severity score with an increase in body weight by suppression of Th17 cells. In the CNS, there was decreased expression of pro-inflammatory cytokine IL-17, an increase in brain infiltrating CD4+ T cells and B cells, while anti-inflammatory phenotype such as FoxP3 was perturbed. Based on preliminary miRNA sequencing data and in silico examination of differential expression of miRNAs, we found that the decrease in neuroinflammation following treatment may be due to up-regulation of miRs, mmu-miR-129–3p, mmu-miR-129-5p, mmu-miR-125-5p and mmu-miR-193-3p as well as down-regulation of mmu-miR-21-3p, mmu-miR-142-5p and mmu-miR147-3p in immune cells in the CNS. Target genes were predicted by TagetScan, miRbase, miRDB. The target genes associated with upregulated miRs included Hrk, CHMP2b, Arid3a and Dcaf7 which are mostly involved in apoptosis, autophagy and migration, while genes associated with down-regulated miRNAs were Abi2, ZFPM2, NDUFA4 that are mostly involved in cell growth, proliferation and differentiation. In summary, Δ-8-THC treatment may offer novel strategies for treatment of neuroinflammation by targeting miRs. Supported by NIH P01AT003961, P20GM103641, R01AI129788, R01 ES030144, R01 AI160896 and R01AI123947

Probing the Mechanism of Action of Cry41Aa on HepG2 through the Establishment of a Resistant Subline.

Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium Bacillus thuringiensis that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin. Concentrations of Cry41Aa that killed over 50% of the parental HepG2 cells had no significant effect on the viability of the resistant cells and did not induce either pore formation or p38 phosphorylation (both characteristic features of pore-forming toxins). Preliminary RNA sequencing data identified AQP9 as a potential mediator of resistance, but extensive investigations failed to show a causal link and did not support an enhanced cell repair process as the resistance mechanism.

MM-associated circular RNA downregulates microRNA-19a through methylation to suppress proliferation of pancreatic adenocarcinoma cells

ABSTRACT Opposite roles of circular RNA MM-associated circular RNA (circ-MYBL2) have been observed in different malignancies, and its role in pancreatic adenocarcinoma (PA) is unknown. Our preliminary sequencing data revealed its inverse correlation with microRNA-19a (miR-19a). This study was performed to explore the role of circ-MYBL2 in PA and its crosstalk with miR-19a. The accumulation of circ-MYBL2 and miR-19a in PA was detected by RT-qPCR. Participation of circ-MYBL2 in the regulation of miR-19a and its RNA gene methylation was studied with an overexpression assay, followed by RT-qPCR and MSP analyses. The role of miR-19a and circ-MYBL2 in PA cell proliferation and movement was evaluated using the BrdU assay and the Transwell assay, respectively. Downregulation of circ-MYBL2 and upregulation of miR-19a were observed in PA. In PA cells, circ-MYBL2 decreased the accumulation of miR-19a but increased its RNA gene methylation. Overexpression of circ-MYBL2 decreased PA cell proliferation and movement, while overexpression of miR-19a showed an opposite effect. In addition, circ-MYBL2 suppressed the role of miR-19a in cell proliferation, migration, and invasion. In conclusion, circ-MYBL2 was downregulated in PA and it downregulated miR-19a through methylation to suppress PA cell proliferation.

Long non-coding RNA Opa interacting protein 5-antisense RNA 1 binds to micorRNA-34a to upregulate oncogenic PD-L1 in non-small cell lung cancer

ABSTRACT Long non-coding RNA (lncRNA) OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) plays an oncogenic role in several types of cancer, but whether it is involved in non-small-cell lung cancer (NSCLC) is unclear. Our preliminary sequencing analysis revealed the upregulation of OIP5-AS1 in NSCLC. In this study, gene expression levels were analyzed by RT-qPCR. RNA-RNA pull-down assay was applied to detect direct interactions between RNAs. Overexpression assays were performed to explore the relationship between miR-34a and OIP5-AS1. CCK-8 assay and colony formation assay were applied to evaluate cell proliferation. In NSCLC cells (H23), overexpression of OIP5-AS1 increased the expression levels of programmed death-ligand 1 (PD-L1). In addition, inhibition of OIP5-AS1 and overexpression of miR-34a decreased the expression levels of PD-L1, and miR-34a significantly blocked the role of overexpression of OIP5-AS1. Overexpression of OIP5-AS1 and PD-L1 promoted H23 and H22 cells proliferation, while silencing of miR-34a and OIP5-AS1 played opposite roles and eliminated the effects of overexpression of OIP5-AS1 on cell proliferation. Therefore, OIP5-AS1 was upregulated to enhance the expression of oncogenic PD-L1 by sponging miR-34a in NSCLC, leading to promoted NSCLC cell proliferation. Our study also demonstrated that OIP5-AS1 was upregulated while miR-34a was downregulated in NSCLC.

Overexpressing circ_0000831 is sufficient to inhibit neuroinflammation and vertigo in cerebral ischemia through a miR-16-5p-dependent mechanism

Circular RNAs (circRNAs) hold potential as stroke-related biomarkers due to involvement in various pathophysiological processes associated with cerebral ischemia and stability in peripheral blood. Differentially expressed circulating circRNAs were identified by preliminary sequencing analysis, through which we identified underexpressed circ_0000831 in ischemic stroke (IS). Validation was performed in peripheral blood of IS patients by quantitative polymerase chain reaction. Microglia was exposed to oxygen-glucose deprivation (OGD), where polarization phenotypes and inflammation were assessed. Middle cerebral artery occlusion was performed in mice to mimic ischemic stroke-induced vertigo, where cerebral blood flow, neurological deficits, vertigo degree, infarct area, inflammation and cell apoptosis were assayed in response to ectopic expression and knockdown of circ_0000831, miR-16-5p, and AdipoR2. Mechanically, circ_0000831 bound to miR-16-5p and downregulated miR-16-5p, and AdipoR2 was targeted by miR-16-5p and increased PPARγ expression in microglia. Furthermore, circ_0000831, AdipoR2, or PPARγ overexpression or miR-16-5p inhibition alleviated neuroinflammation, vertigo, neurological deficit, and cell apoptosis in MCAO mice. Consistently, circ_0000831, AdipoR2, or PPARγ upregulation or miR-16-5p downregulation diminished apoptosis and inflammation of OGD-induced microglia. Consequently, these findings pinpoint the circ_0000831/miR-16-5p/AdipoR2 axis as an essential signaling pathway during ischemia stroke. Thus, the circRNA circ_0000831 may work as a possible target for novel treatment in patients with ischemic stroke.

EP403: Finding merit in impurity: Designing a cost-effective workflow for saliva genome sequencing

Saliva is often the specimen of choice when selecting a non-invasive sample type. Properly collected, saliva is more stable and convenient to ship than blood. However, DNA extracted from saliva is notorious for its bacterial contamination, with bacterial content as high as 50% in some collections. While a high percentage of bacterial DNA may not interfere with many genetic tests, it constitutes an obstacle for genome sequencing (GS) by consuming valuable sequencing space. We aimed at designing and implementing cost-effective processes that would enable us to offer PCR-free GS using saliva-derived DNA as a clinical test. Saliva specimens were provided by volunteers from HudsonAlpha Institute of Biotechnology after written informed consent was obtained. Saliva specimens were collected with Oragene OGD-600 and OGD-675 kits (DNAgenotek). DNA was extracted with the QIAsymphony SP instrument. Libraries for PCR-free GS were generated using 0.5-1.5 μg of saliva-derived DNA with the TruSeq DNA PCR-free kit and IDT for Illumina TruSeq DNA Indices (Illumina). Sequencing was performed using the NovaSeq 6000 System, S4 flowcell, and v1.5 reagent kit. Data processing was performed with an in-house secondary pipeline that utilizes BWA-MEM (via Sentieon) for alignment, strelka2 for variant calling, and kraken2 for measuring percent human DNA versus percent bacterial contamination. Tertiary analysis was performed using an in-house tool, Codicem. Several development and optimization experiments were performed to assess: 1) input requirements for PCR-free GS from saliva-derived DNA; 2) correlation between percent aligned reads and percent human DNA; and 3) genome coverage requirements needed to accurately assess bacterial contamination. We have observed that an input of 0.5 - 1 μg can be used for saliva-derived DNA, consistent with inputs for blood-derived DNA. We also found that an initial low coverage run (1X) for saliva-derived DNA could be used to estimate bacterial contamination of a sample, and our analysis pipeline was able to accurately estimate percent human DNA at a coverage as low as 0.5X. The percent aligned reads differed largely between individuals due to high variability in bacterial DNA contamination. As expected, percent aligned reads correlated with percent human DNA (R2=0.998). The estimation of bacterial content could then be used to facilitate the most efficient sample processing by informing the amount of library required to achieve the desired coverage. We further investigated whether a different collection method of saliva allowed us to improve sequencing efficiency. A sponge collection kit (OGD-675) showed a substantial reduction in bacterial contamination with increase in percent human DNA and percent reads aligned compared to a spit collection kit (OGD-600). On average, human DNA content was 95.44% in sponge kits compared to 85.15% in spit kits. Furthermore, the distribution of percent aligned reads in GS data obtained from saliva specimens collected with sponge kits was much narrower than that for the samples collected with the spit kit (99.46-83.47% compared to 97.09-46.44%). Despite smaller collection volumes in sponge kits and subsequent lower total DNA yields (average of 7.8 μg in sponge kits compared to 12 μg in spit kits), we observed DNA amounts extracted from sponge kits to be sufficient for primary and confirmatory testing. A supplementary stability study performed using DNA isolated 0 and 31 days after collection showed that bacterial contamination did not significantly increase over time for either sponge or spit collection kits (average increase from 3.01% to 3.56% in sponge kits and from 10.27% to 12.31% in spit kits). However, we noted that percentage aligned reads decreased more during the 30 days of storage in samples with high bacterial contamination at initial extraction, with up to 8% increase in bacterial contamination observed for samples with initial human DNA content less than 60%. Consequently, we observed a narrower distribution of percentage aligned read changes over time in DNA isolated from the sponge collection kits (0.01-1.58% compared to 0.1-8.8%). HudsonAlpha Clinical Services Lab has validated PCR-free GS assay with saliva-derived DNA as an input. The workflow efficiency is increased due to the implementation of a preliminary sequencing to 1X coverage as a method to estimate bacterial contamination. This step allows us to triage samples and eliminate failures due to insufficient coverage, as well as balance sequencing pools to achieve desired coverage. As a result of our studies, we found that a sponge saliva collection kit provides sufficient DNA yields with relatively low bacterial contamination, making the sponge kit the preferred collection method for GS applications.

Long non-coding RNA breast cancer-associated transcript 54 sponges microRNA-1269b to suppress the proliferation of hemangioma-derived endothelial cells

ABSTRACT Long non-coding RNA (lncRNA) breast cancer-associated transcript 54 (BRCAT54) and microRNA-1269b (miR-1269b) are two critical ncRNAs in cancer biology, while their roles in hemangioma are unknown. Our preliminary sequencing data revealed their altered expression in hemangioma and predicted they could interact with each other. This study was therefore carried out to investigate the roles of BRCAT54 and miR-1269b in hemangioma, with a focus on their interaction. In this study, hemangioma samples donated by 20 infantile hemangioma patients at proliferating-phase and 20 infantile hemangioma patients at involuting-phase were used. The expression of BRCAT54 and miR-1269b in hemangioma samples, as well as hemangioma-derived endothelial cells (HDECs) and human umbilical vein endothelial cells (HUVECs) were detected by RT-qPCR. IntaRNA 2.0 was applied to predict the interaction between BRCAT54 and miR-1269b, which was then confirmed by RNA-RNA pulldown assay. Accumulation of BRCAT54 in the subcellular location of HDECs was detected by subcellular fractionation assay. The role of BRCAT54 and miR-1269b in cell proliferation has been explored by the BrdU assay. Compared to proliferating-phase tissues, involuting-phase tissues exhibited decreased expression levels of BRCAT54 and increased expression levels of miR-1269b. HDECs had decreased expression levels of BRCAT54 and increased expression levels of miR-1269b compared to that of HUVECs. In HDECs, BRCAT54, which was detected in both nuclear and cytoplasm fractions, directly interacted with miR-1269b. BRCAT54 and miR-1269b did not affect the expression of each other, while BRCAT54 suppressed the role of miR-1269b in enhancing the proliferation of HDECs. BRCAT54 may sponge miR-1269b to suppress the proliferation of HDECs.

Tracing the SARS-CoV-2 infection on the ocular surface: Overview and preliminary corneoscleral transcriptome sequencing

COVID-19's impact on the ocular surface has already been recognized, however the molecular mechanisms induced by the infection on the ocular surface are still unclear. The aim of this paper is to provide a first overview of the transcriptional perturbations caused by SARS-CoV-2 on the ocular surface by analyzing gene expression profile of corneoscleral ring samples from post-mortem SARS-CoV-2 positive donors (PD). The presence of SARS-CoV-2 on the ocular surface, in tears and corneal tissues has rarely been detected in infected individuals in both the presence and the absence of ocular manifestations. In this preliminary study, 6 human corneoscleral tissues of 3 PD and two tissues from a negative donor (CTRL) were obtained at the local eye bank. The presence of genomic and sub-genomic SARS-CoV-2 RNAs was assessed by qRT-PCR, while transcriptome analysis (RNA-sequencing) was performed by Illumina. Principal Component Analysis (PCA), search for differentially expressed genes (DEGs) and Gene Ontology (GO)-enrichment analysis were performed. Three samples from PD were found positive for SARS-CoV-2 genomic RNA, although the absence of sub-genomic RNAs indicated an inactive virus. PCA analysis grouped 3 different clusters, one including CTRL, and the other two including, respectively, PD with undetected SARS-CoV-2 (PD-SARS-neg) and PD with detected SARS-CoV-2 (PD-SARS-pos). The DEGs in common with the 2 PD clusters included several genes associable to the interferon pathway, such as ADAMTS4, RSAD2, MMP1, IL6, ISG15 and proinflammatory cytokines. Among the down-regulated genes we found AQP5. GO analysis revealed 77 GO terms over-represented in PD-SARS-neg vs. CTRL, and 17 GO terms in PD-SARS-pos vs. CTRL.The presence of SARS-CoV-2 RNA and RNA-sequencing reads in ocular surface tissues supports the possibility that the eye acts as an entry route. The modulation of early responsive genes, together with several ISGs suggests a potential protective responsiveness of the ocular tissues to SARS-CoV-2.

New and interesting species of Penicillium (Eurotiomycetes, Aspergillaceae) in freshwater sediments from Spain.

Penicillium species are common fungi found worldwide from diverse substrates, including soil, plant debris, food products and air. Their diversity in aquatic environments is still underexplored. With the aim to explore the fungal diversity in Spanish freshwater sediments, numerous Penicillium strains were isolated using various culture-dependent techniques. A preliminary sequence analysis of the β-tubulin (tub2) gene marker allowed us to identify several interesting species of Penicillium, which were later characterized phylogenetically with the barcodes recommended for species delimitation in the genus. Based on the multi-locus phylogeny of the internal transcribed spacer region (ITS) of the ribosomal DNA, and partial fragments of tub2, calmodulin (cmdA), and the RNA polymerase II largest subunit (rpb2) genes, in combination with phenotypic analyses, five novel species are described. These are P.ausonanum in sectionLanata-Divaricata, P.guarroi in sect.Gracilenta, P.irregulare in sect.Canescentia, P.sicoris in sect.Paradoxa and P.submersum in sect.Robsamsonia. The study of several isolates from samples collected in different locations resulted in the reinstatement of P.vaccaeorum into sectionCitrina. Finally, P.heteromorphum (sect.Exilicaulis) and P.tardochrysogenum (sect.Chrysogena) are reported, previously only known from Antarctica and China, respectively.

LncRNA LOXL1-AS1 inhibits proliferation of PDLSCs and downregulates IL-1β in periodontitis patients.

The carcinogenic role of lncRNA LOXL1-AS1 in thoracic aortic aneurysm has been confirmed, but its role in the pathogenesis of periodontitis remains unclear. Our preliminary deep sequencing data revealed LOXL1-AS1 downregulation in periodontitis and its inverse correlation with IL-1β, a critical inflammatory mediator in periodontitis. This study was therefore performed to investigate the potential interaction between LOXL1-AS1 and IL-1β in periodontitis. The study included 30 periodontitis patients (18males and 12 females at the age of 34 to 44years, with a mean of 39.3±2.1years) and 30healthy controls (18males and 12 females at the age of 33 to 44years with a mean of 39.2±2.0years). The effects of LOXL1-AS1 overexpression on IL-1β were evaluated by RT-qPCR and Western blot. CCK-8 assay was used to analyze cell proliferation. LOXL1-AS1 was downregulated in periodontitis-affected periodontal ligament stem cells (PDLSCs) compared with healthy PDLSCs, while IL-1β was upregulated in periodontitis-affected PDLSCs and was inversely correlated with LOXL1-AS1. LOXL1-AS1 overexpression mediated IL-1β downregulation in PDLSCs. IL-1β treatment did not affect LOXL1-AS1 expression. Moreover, LOXL1-AS1 overexpression inhibited the proliferation of periodontitis-affected PDLSCs. LOXL1-AS1 overexpression and IL-1β knockdown increased Bax/Bcl-2 ratio and caspase-3level. This study is the first to report LOXL1-AS1 downregulation in periodontitis. Moreover, LOXL1-AS1might inhibit the proliferation of periodontitis-affected PDLSCs and downregulate IL-1β to improve periodontitis.

New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene.

ABSTRACTThe identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes.IMPORTANCE Legionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non-pneumophila species identification both in clinical and environmental samples. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies’ MinION technology. This enabled ‘proof of concept’ for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32–07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32–07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

LncRNA-TUG1 Downregulation is Correlated with the Development of Progressive Chronic Kidney Disease Among Patients with Congestive Heart Failure.

TUG1 is an lncRNA that plays a critical role in kidney injury. Our preliminary sequencing analysis showed altered TUG1 expression in both congestive heart failure (CHF) patients and CHF patients complicated with chronic kidney disease (CKD). We then studied the role of TUG1 in these two diseases. We enrolled 120 CHF patients without obvious complications, 60 CHF patients complicated with CKD, and 60 healthy volunteers. TUG1 expression in plasma samples from these participants was determined using RT-qPCR. The diagnostic value of TUG1 for CKD development in CHF patients was evaluated by ROC curve analysis. A 2-year follow-up was performed to detect the correlation between TUG1 expression levels and the development of CKD in CHF. TUG1 was lowly expressed in CHF patients and was further downregulated in CHF patients complicated with progressive CKD. ROC curve analysis showed that plasma TUG1 expression levels could be used to distinguish CHF patients complicated with CKD from CHF patients without CKD and healthy controls. During the 2-year follow-up, high CHF expression levels predicted a low incidence of progressive CKD among CHF patients. With the treatment of progressive CHF + CKD, plasma TUG1 was upregulated. LncRNA-TUG1 downregulation may develop the progressive CKD among patients with CHF.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis.

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

Metagenomic Investigation of a Low Diversity, High Salinity Offshore Oil Reservoir.

Oil reservoirs can represent extreme environments for microbial life due to low water availability, high salinity, high pressure and naturally occurring radionuclides. This study investigated the microbiome of saline formation water samples from a Gulf of Mexico oil reservoir. Metagenomic analysis and associated anaerobic enrichment cultures enabled investigations into metabolic potential for microbial activity and persistence in this environment given its high salinity (4.5%) and low nutrient availability. Preliminary 16S rRNA gene amplicon sequencing revealed very low microbial diversity. Accordingly, deep shotgun sequencing resulted in nine metagenome-assembled genomes (MAGs), including members of novel lineages QPJE01 (genus level) within the Halanaerobiaceae, and BM520 (family level) within the Bacteroidales. Genomes of the nine organisms included respiratory pathways such as nitrate reduction (in Arhodomonas, Flexistipes, Geotoga and Marinobacter MAGs) and thiosulfate reduction (in Arhodomonas, Flexistipes and Geotoga MAGs). Genomic evidence for adaptation to high salinity, withstanding radioactivity, and metal acquisition was also observed in different MAGs, possibly explaining their occurrence in this extreme habitat. Other metabolic features included the potential for quorum sensing and biofilm formation, and genes for forming endospores in some cases. Understanding the microbiomes of deep biosphere environments sheds light on the capabilities of uncultivated subsurface microorganisms and their potential roles in subsurface settings, including during oil recovery operations.