Fluorescent PCR Research Articles

Clinicopathological features of verrucous hemangioma

Objective: To investigate the clinicopathological features, and differential diagnosis of verrucous hemangioma (VH). Methods: Twenty-eight VH cases diagnosed from 2005 to 2020 in Henan Provincial People's Hospital, Zhengzhou, China were analyzed retrospectively. Immunohistochemical studies were used to detect diagnostic markers. The mutation status of PIK3CA (exons 9 and 20) was detected using fluorescence PCR. Results: There were 13 males and 15 females in 28 cases, with the male to female ratio of 1.0∶1.2. There were 25 patients under the age of 18 years. The age range was from 10 months to 56 years (mean, 9.7 years; median, 4.5 years). There were 17 cases occurred in the lower extremities, 7 in the upper extremities and 4 in the trunk. All 28 cases were irregular red patches on the skin, which grew slowly. Some of them were thickened with uneven surface, which was light pink or red-white. Skin lesions of the 7 cases ranged from dark red and reddish brown, with a rough and hard surface. Satellite foci were present. Microscopically, 28 cases had a wide range of pathological features. Dilated, malformed vessels were observed from dermal papilla to deep soft tissue. Among them, the dermal papillary layer was mainly composed of many proliferating and expanding thin-walled capillaries and cavernous blood vessels. Thin-walled small vessels were found in the dermal reticular layer and subcutaneous fascia layer, with no obvious endothelial cell proliferation, occasional papillary hyperplasia, and lobular distribution of the malformed vessels in the fascia layer mixed with the fibroadipose tissue. There was epidermal papillary hyperplasia with hyperkeratosis and parakeratosis, lengthening and mutual fusion of epithelial horns. Immunohistochemistry showed that CD31, CD34, ERG and WT-1 were diffusely and strongly positive. The expression of GLUT-1 was present in superficial dermal vascular endothelial cells, but undetectable in the deep layer. The PIK3CA tests of 13 cases showed that no somatic mutations were found in exons 9 and 20. Twenty-five patients were followed up for 5 months to 10 years. Seven patients underwent multiple surgical resections and plastic surgeries due to the large size, and 8 patients had recurrence. Conclusions: VH is a rare congenital vascular malformation and more commonly occurs in infants and children. It tends to appear in limbs, especially lower limbs and distal limbs. Its morphology and immunophenotype are characteristic and should be distinguished from other vascular malformations and the resolution phase of infant hemangiomas. In about one third of the cases, postoperative recurrence may occur and long-term follow-up is often required.

Depth effects on bacterial community assembly processes in paddy soils

Bacterial communities in soil play a key role in carbon (C) and nutrient cycling. Unravelling how bacterial community assemble and distribute with soil depth is a prerequisite for understanding microbial functions, nutrient cycling and management. Twenty-six rice fields in a typical red soil area in a wet subtropical climate were sampled in the topsoil (0–10 and 10–20 cm) and subsoil (20–40 cm). Physico-chemical soil properties, quantitative fluorescence PCR and high-throughput sequencing were used to analyse the V4 region of 16S rDNA. The rRNA operon copy number and alpha diversity decreased continuously with soil depth because of reduced access to carbon, energy, oxygen and nutrients. The relative abundance of the dominant phyla Proteobacteria and Actinobacteria decreased with increasing soil depth, whereas the opposite trend was observed for the phylum Nitrospirae. The interaction intensity between taxa increased with depth, as limited carbon and nutrients in the undisturbed subsoil lead to the cooccurrence of taxa with similar ecological niches that cooperated to reduce functional redundancy. The higher modularity of the bacterial network in the topsoil is associated with greater environmental perturbations (flooding, fertilization, etc.) to maintain the robustness of the microbial community. Bacterial community assembly processes were stochastic up to 40 cm, but ecological drift was the predominant process in the topsoil, whereas dispersal limitation was dominant in the subsoil. The contribution of abiotic factors (e.g. nutrient and iron contents) and biotic factors (taxa-taxa interactions) as well as dispersal limitations to bacterial community assembly was depth specific. Concluding, the basic principles of bacterial community assembly were evaluated for the first time for a broad range of paddy soils.

18. Global Surveillance of Clostridioides difficile Demonstrates High Prevalence in Non-Healthcare Settings

Background Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing organism that is the leading cause of healthcare-associated infections. However, past studies have isolated C. difficile spores from the community, suggesting an environmental reservoir that may play a role in transmission. This study aimed to examine the prevalence and strain types of C. difficile isolated from the United States (US) and internationally.MethodsFrom 2014 to 2017, environmental swabs were collected from public areas, healthcare settings, and shoe soles. Samples were considered positive for C. difficile following growth on CCFA plates and confirmatory PCR testing for toxin genes and fluorescent PCR ribotyping (RT). The rate of C. difficile positivity and associated RT distribution were compared between settings, including shoe soles which were investigated for their potential role in environmental transmission.ResultsA total of 11,986 unique isolates were obtained primarily from the US (n=11,002; 92%) in addition to 11 other countries including Taiwan (n=200) and India (n=187). Samples were categorized as being from outdoor environments (n=2,992), private residences (n=2,772), shoe soles (n=1,420), public buildings (n=1,104) or acute care settings (n=3,698). Worldwide C. difficile sample positivity was 26% and was similar between US and non-US sampling sites. In the US, private residences (26.2%) and outdoor environments (24.1%) had the highest positivity rate compared to public buildings (17.2%). In a Texas sub-analysis (n=8,571), positivity rates were highest from outdoor samples (27%) and were similar between private residences (24%) and healthcare buildings (24%). The most prevalent RTs overall were F014-020 (16.4%), F106 (14.9%), and FP310 (11%). Shoe soles had the highest positivity rate (45%) with similar RT distribution between shoe soles and environmental samples.ConclusionUsing a worldwide sample, 26% of environmental samples tested positive for toxigenic C. difficile strains from healthcare and non-healthcare sites. Community stewardship efforts will be needed to reduce the risk of CDI in vulnerable patients. Shoe sole sampling may be an ideal surveillance tool to test for emerging epidemic strains.Disclosures Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support)

Brucella abortus S19 GFP-tagged vaccine allows the serological identification of vaccinated cattle.

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis.

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.

Abstract 12777: Bmi1 Mediates Chromatin Remodeling and Redox Homeostasis for Cardiac Repair After Myocardial Injury

Introduction: Myocardial injury leads to oxidative stress that has a significant impact on the development and progression of disease. Increasing evidence suggests that oxidative stress globally influences chromatin landscape of the cells. Effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including DNA damage, cell death, and accelerated senescence which are all disease-driving mechanisms during the failing heart. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment after myocardial injury. The Polycomb complex protein BMI-1 (Bmi1) an epigenetic regulator, is associated with numerous biological functions including, mediating DNA damage control, ROS signaling and senescence. However, there is currently a lack of understanding on how Bmi1 mediates epigenetic modifications affecting heart function during injury and is a focus of this study. Hypothesis: The epigenetic regulation by Bmi1 mediates reparative process after myocardial injury by regulating ROS production. Specifically, Bmi1 modulates the epigenetic landscape of cardiac cells that mediates various molecular processes during development and stress conditions. Methods and Results: Using a Bmi1 knockout (KO) model through the induction of a tamoxifen Cre recombinase system, cardiac function is determined through echocardiography using adult mice following myocardial infarction. The Bmi1 KO has a significant decrease in heart function after myocardial infarction, which has been associated with increased cellular ROS production, fibrosis, and apoptotic mechanisms which are measured by fluorescent ROS assays, TUNEL assays, western blotting, PCR, and immunohistochemistry. To date, we have found that cardiac cells, including adult cardiomyocytes and adult cardiac fibroblasts, isolated from the Bmi1 KO model have increased cellular ROS production, increased expression of fibrotic genes including Acta2, Vimentin, Collagen, and increased expression of apoptotic pathway regulators including Aldo1, Eno1, NFkB, Ap-1, and HIF1-a. Conclusions: Bmi-1 regulation mediates repair during pathological challenge by regulating ROS production after myocardial injury.

Urbanization Affects Soil Microbiome Profile Distribution in the Russian Arctic Region.

Urbanization in the Arctic results in considerable and still poorly known environmental consequences. The effect of urbanization on soil microbiome—an ecosystem component highly sensitive to anthropogenic disturbance—remains overlooked for the Arctic region. The research compared chemical and microbial properties of the natural Podzol soils and urban soils of Murmansk—the largest Arctic city. Particular attention was given to the profile distribution, which is almost completely ignored by most microbial studies. Soil microbiome was investigated by the quantitative indicators based on fluorescence microscopy (microbial biomass) and PCR real-time methods (amount of rRNA genes copies of archaea, bacteria, and fungi). The principal changes in urban soils’ properties compared to the natural references included a shift in pH and an increase in C and nutrients’ contents, especially remarkable for the subsoil. The numbers of rRNA genes copies of archaea, bacteria, and fungi in urban topsoils (106–1010, 109–1010, and 107–109, respectively) were lower than in Podzol; however, the opposite pattern was shown for the subsoil. Similarly, the total microbial biomass in urban topsoils (0.55–0.75 mg g−1) was lower compared to the 1.02 mg g−1 in Podzols, while urban subsoil microbial biomass was 2–2.5 times higher than in the natural conditions. Both for urban and natural soils and throughout the profiles, fungi were dominated by mycelium forms; however, the ratios of mycelium–spores were lower, and the amount of thin mycelium was higher in urban soils than in natural Podzols. Urbanization in the Arctic altered soil morphological and chemical properties and created a new niche for microbial development in urban subsoils; its contribution to biodiversity and nutrient cycling promises to become increasingly important under projected climate change.

Correlation of conventional ultrasound features and related factors with BRAFV600E gene mutation in papillary thyroid carcinoma

Objective:To investigate the correlation of conventional ultrasound features and related factors with BRAFV600E gene mutation in papillary thyroid carcinoma（PTC）. Methods:A total of 266 nodules in 208 patients with PTC confirmed by operation and pathology in First Affiliated Hospital of Jinzhou Medical University from October 2019 to March 2021 were selected. BRAFV600E gene was detected by real-time fluorescence PCR quantitative technique after operation. According to the results of gene detection, the patients were divided into gene mutation positive group and gene mutation negative group. The routine ultrasound features and clinical data of the patients were collected, and the correlation with BRAFV600E gene mutation was analyzed. Results:Among the 266 samples, 188 cases（70.7%） had gene mutation. Univariate analysis showed that there were significant differences in age, nodular edge, capsule boundary, microcalcification and abnormal cervical lymph nodes between the two groups（P<0.05）. The results of Logistic multivariate regression analysis showed that age, nodular margin and abnormal cervical lymph nodes were independent influencing factors of BRAFV600E gene mutation in patients with PTC. ROC curve showed that when the age was 46.5 years old, the Youden index was the highest. Conclusion:BRAFV600E gene mutation is associated with age≤46.5 years old, uneven edge of nodules and abnormal lymph nodes in neck in patients with PTC. The above characteristics can be used to evaluate BRAFV600E gene mutation and have certain guiding significance for clinical diagnosis, treatment and prognosis.

Comparison of Lp-PLA2 Activity, Lp-PLA2 Mass and Lp-PLA2 mRNA in Acute Coronary Syndrome Patients

Background: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk predictor for cardiovascular diseases (CVD). Generally, plasma Lp-PLA2 was thought to be secreted by circulatory inflammatory cells. Lp-PLA2 mRNA expression of PBMC may also be a risk predictor. Methods: A total of 104 subjects angiographically verified ACS patients were enrolled, including 73 unstable angina pectoris (UAP) patients and 31 acute myocardial infarction (AMI) patients. Plasma lipids, Lp-PLA2 activity and Lp-PLA2 mass were measured. Lp-PLA2 mRNA expression of PBMC was relatively quantified by real-time fluorescence PCR. Results: Plasma Lp-PLA2 activity was increased in AMI patients compared to UAP patients (395.21±145.91 vs. 328.53±127.03 U/L, p=0.024). Lp-PLA2 mass of AMI patients was also higher than UAP patients (136.43±45.46 vs. 119.16±44.19 ng/mL, p=0.093), while PBMC mRNA expression was not statistically different [1.07 (0.74, 1.57) vs. 0.88(0.49, 1.99), p=0.453]. Comparing Lp-PLA2 mRNA by groups, Lp-PLA2 mRNA level was higher in male ACS patients and smoking ACS patients (p=0.008, p=0.048, respectively). Multivariate logistic regression analysis showed that Lp-PLA2 activity was an AMI risk predictor (OR=5.224, 95% CI 1.687-16.181, p=0.004), after smoking, systolic blood pressure, diabetes and hyperlipidemia were adjusted. Recurrent ACS patients were older (p=0.035), but they showed lower levels of Lp-PLA2 mass and Lp-PLA2 activity (p=0.014, p=0.045, respectively), compared to primary ACS patients. Conclusion: Smoking may be an important regulatory factor for Lp-PLA2 mRNA expression in PBMC. Among three Lp-PLA2 indexes, Lp-PLA2 activity was the best marker indicating AMI risk, while Lp-PLA2 mass maybe play better role as a predictor in avoiding ACS recurrence.

A Coagulation-Related Gene-Based Prognostic Model for Invasive Ductal Carcinoma

Background: Invasive ductal carcinoma (IDC) is the most common type of metastatic breast cancer. Due to the lack of valuable molecular biomarkers, the diagnosis and prognosis of IDC remain a challenge. A large number of studies have confirmed that coagulation is positively correlated with angiogenesis-related factors in metastatic breast cancer. Therefore, the purpose of this study was to construct a COAGULATION-related genes signature for IDC using the bioinformatics approaches.Methods: The 50 hallmark gene sets were obtained from the molecular signature database (MsigDB) to conduct Gene Set Variation Analysis (GSVA). Gene Set Enrichment Analysis (GSEA) was applied to analyze the enrichment of HALLMARK_COAGULATION. The COAGULATION-related genes were extracted from the gene set. Then, Limma Package was used to identify the differentially expressed COAGULATION-related genes (DECGs) between ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) samples in GSE26340 data set. A total of 740 IDC samples from The Cancer Genome Atlas (TCGA) database were divided into a training set and a validation set (7:3). The univariate and multivariate Cox regression analyses were performed to construct a risk signature, which divided the IDC samples into the high- and low-risk groups. The overall survival (OS) curve and receiver operating characteristic (ROC) curve were drawn in both training set and validation set. Finally, a nomogram was constructed to predict the 1-, 2-, 3-, 4-, and 5-year survival rates of IDC patients. Quantitative real-time fluorescence PCR (qRT-PCR) was performed to verify the expression levels of the prognostic genes.Results: The “HALLMARK_COAGULATION” was significantly activated in IDC. There was a significant difference in the clinicopathological parameters between the DCIS and IDC patients. Twenty-four DECGs were identified, of which five genes (SERPINA1, CAPN2, HMGCS2, MMP7, and PLAT) were screened to construct the prognostic model. The high-risk group showed a significantly lower survival rate than the low-risk group both in the training set and validation set (p=3.5943e-06 and p=0.014243). The risk score was demonstrated to be an independent predictor of IDC prognosis. A nomogram including risk score, pathological_stage, and pathological_N provided a quantitative method to predict the survival probability of 1-, 2-, 3-, 4-, and 5-year in IDC patients. The results of decision curve analysis (DCA) further demonstrated that the nomogram had a high potential for clinical utility.Conclusion: This study established a COAGULATION-related gene signature and showed its prognostic value in IDC through a comprehensive bioinformatics analysis, which may provide a potential new prognostic mean for patients with IDC.

"HLA-10-SNP": A new qPCR-based system for rapid, effective, and accurate detection of 10 important SNPs in the HLA region.

Mastering the SNP content in the HLA region can be based on it to judiciously select unrelated donor stem cells with preferable MHC matching to lower postoperative complications. Herein, quantitative PCR-based primers and probes were designed for 10 transplants outcome-associated SNP loci with two-allelic polymorphism, and then a new detection system ("HLA-10-SNP") was established. Compared with Sanger sequencing, its accuracy has been proven to reach 100%. Additionally, fluorescent PCR typing of 10 important SNPs via this system expressed excellent repeatability (sensitivity, 20 ng). Overall, the new system achieves single-sample classification precision and easily distinguishable results, equipped with the advantages of simple, rapid, accurate, and effective, promising to acquire widespread popularization and application in clinical settings.

Effect of CAG repeats on the age at onset of patients with spinocerebellar ataxia type 2 in China.

Spinocerebellar ataxia type 2 (SCA2) is one of the most common autosomal dominant ataxias in the world. Several reports revealed that CAG repeats in some polyQ-containing genes may affect the age at onset (AAO) of patients with SCA2, however, little studies were conducted among Chinese patients with SCA2. Thus, the aim of this study is to evaluate the effect of CAG repeats on the AAO of patients with SCA2 in China. A total of 119 patients with SCA2 were enrolled and were divided into 2 groups according to their major phenotype: 17 patients from 9 families with Parkinson's syndrome were grouped as the Parkinson's disease-SCA2 (PD-SAC2); 91 patients from 66 SCA2 families and 11 sporadic SCA2 patients were grouped as the ataxia-SCA2 (A-SCA2). Blood samples were obtained from the subjects, and the CAG repeat length in ATXN2 and other (CAG)n-containing genes was screened using fluorescent PCR. The Spearman's rank correlation between the CAG repeat length in (CAG)n-containing genes and AAO was analyzed. Regression analysis was performed to investigate whether the CAG repeat length could explain the variant of AAO. A t-test was used to compare the difference of CAG repeat length in (CAG)n-containing genes between the PD-SAC2 and A-SCA2 groups. The CAG repeat length in the longer allele of ATXN2 was negatively correlated with AAO of SCA2 (R=-0.251, P<0.05), and the CAG repeat length could explain 41.7% of the variation of AAO. AAO negatively correlated with the CAG repeat length in the shorter allele of ATXN7 (R=-0.251, P=0.006) or in the longer allele of TBP gene (R=-0.197, P=0.034). A tendency of delay in the AAO was also observed in patients with SCA2 carrying the CAG repeat within the ATXN3, CACNA1A, ATXN7, TBP, and RAI1. In addition, we found that the CAG repeat length in ATXN7 and ATXN2 between the A-SCA2 and the PD-SCA2 groups was significantly different (both P<0.05). The CAG repeat in ATXN2 is a major genetic factor for the AAO of patients with SCA2 in China. The CAG repeat length in ATXN3, CACNA1A, ATXN7, TBP, and RAI1 genes might be a potential factor associated with the AAO of SCA2. The CAG repeat in ATXN7 might be a potential factor affecting the Parkinson's syndrome in SCA2.

Mechanisms Underlying the Protective Effect of the Peroxiredoxin-6 Are Mediated via the Protection of Astrocytes during Ischemia/Reoxygenation.

Ischemia-like conditions reflect almost the entire spectrum of events that occur during cerebral ischemia, including the induction of oxidative stress, Ca2+ overload, glutamate excitotoxicity, and activation of necrosis and apoptosis in brain cells. Mechanisms for the protective effects of the antioxidant enzyme peroxiredoxin-6 (Prx-6) on hippocampal cells during oxygen-glucose deprivation/reoxygenation (OGD/R) were investigated. Using the methods of fluorescence microscopy, inhibitory analysis, vitality tests and PCR, it was shown that 24-h incubation of mixed hippocampal cell cultures with Prx-6 does not affect the generation of a reversible phase of a OGD-induced rise in Ca2+ ions in cytosol ([Ca2+]i), but inhibits a global increase in [Ca2+]i in astrocytes completely and in neurons by 70%. In addition, after 40 min of OGD, cell necrosis is suppressed, especially in the astrocyte population. This effect is associated with the complex action of Prx-6 on neuroglial networks. As an antioxidant, Prx-6 has a more pronounced and astrocyte-directed effect, compared to the exogenous antioxidant vitamin E (Vit E). Prx-6 inhibits ROS production in mitochondria by increasing the antioxidant capacity of cells and altering the expression of genes encoding redox status proteins. Due to the close bond between [Ca2+]i and intracellular ROS, this effect of Prx-6 is one of its protective mechanisms. Moreover, Prx-6 effectively suppresses not only necrosis, but also apoptosis during OGD and reoxygenation. Incubation with Prx-6 leads to activation of the basic expression of genes encoding protective kinases—PI3K, CaMKII, PKC, anti-apoptotic proteins—Stat3 and Bcl-2, while inhibiting the expression of signaling kinases and factors involved in apoptosis activation—Ikk, Src, NF-κb, Caspase-3, p53, Fas, etc. This effect on the basic expression of the genome leads to the cell preconditions, which is expressed in the inhibition of caspase-3 during OGD/reoxygenation. A significant effect of Prx-6 is directed on suppression of the level of pro-inflammatory cytokine IL-1β and factor TNFα, as well as genes encoding NMDA- and kainate receptor subunits, which was established for the first time for this antioxidant enzyme. The protective effect of Prx-6 is due to its antioxidant properties, since mutant Prx-6 (mutPrx-6, Prx6-C47S) leads to polar opposite effects, contributing to oxidative stress, activation of apoptosis and cell death through receptor action on TLR4.

Rapid start of high-concentration denitrification and desulfurization reactors by heterotrophic denitrification sulphur-oxidising bacteria

High sulphide concentrations can be toxic to denitrifying and desulphurising microorganisms. In this study, bioaugmentation was used to solve this problem. Pseudomonas sp. gs1 can tolerate 400 mg/L sulphide and converts most of the sulphide into elemental sulphur after 4 h. A solid inoculum of Pseudomonas sp. h1 was prepared. Two reactors, that is, one with and one without inoculum, were simultaneously run for 60 days. Bioreactor II to which bacterial inoculum was added reached a good treatment performance on day 3. The elemental sulphur concentration of the effluent was 342.6 mg/L. It was maintained at 245.3–333.8 mg/L during the subsequent operation. In contrast, reactor I without inoculants achieved the same performance on day 50. High-throughput sequencing shows that Pseudomonas and Azoarcus are the dominant genera. The abundance of the genus Pseudomonas and related denitrifying sulphur-oxidising bacteria in reactor I increases with the operation time. This phenomenon was confirmed by testing the sqr and gltA genes. The quantitative fluorescence PCR test also proves that the addition of bacteria leads to a rapid increase in the sulphur oxidation and carbon metabolism of the activated sludge in the reactor.

Bioinformatics analysis and function prediction of ginseng TCP transcription factor family

The longevity mechanism of ginseng(Panax ginseng) is related to its strong meristematic ability. In this paper, this study used bioinformatic methods to identify the members of the ginseng TCP gene family in the whole genome and analyzed their sequence characteristics. Then, quantitative real-time fluorescent PCR was performed to analyze the TCP genes containing elements rela-ted to meristem expression in the taproots, fibrous roots, stems, and leaves. According to the data, this study further explored the expression specificity of TCP genes in ginseng tissues, which facilitated the dissection of the longevity mechanism of ginseng. The ginseng TCP members were identified and analyzed using PlantTFDB, ExPASy, MEME, PLANTCARE, TBtools, MEGA and DNAMAN. The results demonstrated that there were 60 TCP gene family members in ginseng, and they could be divided into two classes: Class Ⅰ and Class Ⅱ, in which the Class Ⅱ possessed two subclasses: CYC-TCP and CIN-TCP. The deduced TCP proteins in ginseng had the length of 128-793 aa, the isoelectric point of 4.49-9.84 and the relative molecular mass of 14.2-89.3 kDa. They all contained the basic helix-loop-helix(bHLH) domain. There are a variety of stress response-related cis-acting elements in the promoter regions of ginseng TCP genes, and PgTCP20-PgTCP24 contained the elements associated with meristematic expression. The transcription levels of PgTCP20-PgTCP24 were high in fibrous roots and leaves, but low in stems, indicating the tissue-specific expression of ginseng TCP genes. The Class Ⅰ TCP members which contained PgTCP20-PgTCP23, may be important regulators for the growth and development of ginseng roots.

Tetrasomy 18p in one non-identical twin born to healthy parents: A case report

Tetrasomy 18p is an extremely rare disorder with estimated incidence 1:140,000-180,000. The most common clinical presentations include developmental delay, intellectual disability, and several dysmorphic features such as the triangular face, low-set ears, depressed nasal bridge or smooth philtrum. The disease's hallmark is a small supernumerary isochromosome 18p consisting of two copies of the same arm of chromosome 18. Males and females are affected equally. Here, we present the case of 20 months old girl with tetrasomy 18p, the only affected child out of the dizygotic twins born to healthy parents. Routine cytogenetic testing revealed the presence of the marker chromosome in the girl's sample. Combination of Multicolor Banding fluorescent in situ hybridization and array-based Comparative Genomic Hybridization confirmed the isochromosome 18p in this girl. Quantitative Fluorescence PCR determined the maternal origin of the isochromosome 18p. The presented case of tetrasomy18p in one of the non-identical twins supports the hypothesis that tetrasomy 18p arises de novo and are of maternal origin. We conclude that the formation of isochromosome 18p is a random event that can occur in pregnancies of cytogenetically normal parents and maternal age is probably the main risk factor in etiology of the tetrasomy 18p.

Vaccarin enhances intestinal barrier function in type 2 diabetic mice

Hyperglycemia and insulin resistance drive intestinal barrier dysfunction in type 2 diabetes (T2DM). Vaccarin, the main active component in the semen of traditional Chinese medicine Vaccaria has a definite effect on T2DM mice. The purpose of this study was to investigate whether vaccarin can enhance the intestinal barrier function in T2DM. The T2DM mice model was established by streptozocin and high-fat diet. Vaccarin at a dose of 1mg/kg/day was administered. We evaluated the effects of vaccarin on gut microbiota and intestinal barrier function by 16S rRNA sequencing, Western blot, quantitative fluorescent PCR (qPCR), and morphological observation. Moreover, we constructed a single layer of the human intestinal epithelium model to determine the effect of vaccarin in vitro. The experimental results showed that vaccarin alleviated inflammatory mediators in serum and intestinal tissue of mice (P<0.05), which may depend on the improvement of tight junctions and gut microbiota (P<0.05). Activation of extracellular regulated protein kinases (Erk1/2) stimulated myosin light chain kinase (MLCK). By inhibiting ERK expression (P<0.05), vaccarin had similar effects to ERK inhibitors. In addition, the regulation of tight junction barriers also involved the abovementioned pathways in vivo. Vaccarin could protect the intestinal barrier by inhibiting the ERK/MLCK signaling pathway and modulate the composition of the microbiota. These results suggested that vaccarin may be an effective candidate for improving intestinal barrier changes in T2DM.

Analysis of gut microbiota and immune-related genes during sea cucumber (Apostichopus japonicus) response to dietary supplementation with Codonopsis pilosula

The gut microbiota composition of sea cucumber (Apostichopus japonicas) was investigated using high-throughput sequencing techniques. The mRNA expression of complement component 3 and lysozyme genes was evaluated using quantitative fluorescence PCR. Sea cucumbers were fed with a basal diet (control group) and an experimental diet supplemented with Codonopsis pilosula (experimental group) for 30 days. The results showed that the alpha diversity of the gut microbiota was changed in different indices, including Chao1, the abundance-based coverage estimator, the Shannon index, and Good’s coverage. Dietary C. pilosula promoted the proliferation of the Flavobacteriaceae family of the Proteobacteria phylum and reduced the relative abundance of the Verrucomicrobiaceae family of the Verrucomicrobia phylum. We concluded that dietary C. pilosula supplementation could alter the network interactions among different microbial functional groups by changing the ecological network’s microbial community composition and biological evolution. A positive effect on A. japonicus immune responses in the gut was seen via increasing the mRNA expression of the complement component 3 and lysozyme genes. It seems to happen via modulating the balance in gut microbiota.

Role Of Stress Responsive Genes Responsible In Molecular Switch From Adaptive Phase To Apoptosis In HbE−β Thalassemia

Hemoglobin E (HbE) interacts with different forms of β thalassemia resulting in hemoglobin E-β thalassemia (HbE-β thal) with a heterogeneous clinical phenotype. Enhanced apoptosis is a key feature of ineffective erythropoiesis in β-thalassemia or HbE-β thalassemic erythroid precursors. Heme-regulated eIF2α kinase (HRI) plays an ameliorating role in β-thalassemia intermedia mice by regulating the translation of heme and globin. Extensive studies in mice have shown that absence of Hri-activated Atf4 signalling delays erythroid maturation especially under stress conditions (Chen et al, 2012). Selective translation of ATF4 (Active transcription factor) under stress conditions, in turn, up-regulates pro-apoptotic transcription factor CHOP(C/EBP homologous protein-10), X-box binding protein -1 (XBP1) and stress inducible GADD34 (Growth arrest and DNA damage-inducible protein) which is found to be necessary for the adaptation to stress conditions. Since the mechanism by which apoptosis is induced remains unclear, we aimed to study the role of HRI signalling pathway in ineffective erythropoiesis in HbE-β thalassemia patients. After informed consent, peripheral blood samples were collected from 10 normal individuals, 30 HbE-β thal and 6 homozygous HbE patients. The grading of β-thalassemia/Hb E severity was done based on scoring system as described previously. Reticulocytes were isolated using cellulose columns and peripheral blood mononuclear cells from patients were cultured using a two-phase liquid culture protocol, as described by Fibach,E et al., 1989. Erythroid precursor cells were collected on day 5 (Early stage), day 10 (Intermediate stage) and day15 (Late stage) in phase II culture. Total RNA was extracted using Trizol, followed by cDNA synthesis. Quantitative real time PCR (qRT-PCR) was performed to analyse the expression of HRI, GADD34, CHOP, and ATF4 and relative expression was calculated by 2-DDCt method. We also developed and validated a fluorescent PCR–based analysis (sensitivity-2%) for the detection of unspliced (XBP1U) and spliced (XBP1S) form of XBP1 mRNA and the data was analysed using Genemapper software. Median HRI expression was higher in the reticulocytes obtained from patients with HbE-βthal [1.7(0.10-12)] as compared to controls [1.07 (0.05-4.39)] and a significant up regulation of downstream genes ATF4 and CHOP was observed in HbE-β when compared with controls (Table 1). HRI expression correlated positively with the expression of CHOP(r=0.511; p=0.030), ATF4 (r=0.517; p=0.028) and GADD34 (r=0.72; p=0.000).The ratio of spliced and un-spliced forms of XBP1 did not differ significantly between control and HbE- βthal (Figure 1). Similarly, significant upregulation of HRI (p=0.059) and its downstream genes was also observed on day 15 erythroid precursor (Table 1). We also compared the reticulocyte HRI levels in 15 patients with severe phenotype and 12 with milder phenotype. Significant higher expression of HRI was observed in the patients with milder phenotype [2.89 (0.85 -12.0)] as compared to severe [1.12 (0.1-4.23)] (p=0.043). There was no difference in expression pattern of other genes between mild and severe phenotypes. Reticulocytes of Homozygous HbE shows significant up regulation of ATF4 (p=.012) when compared with controls but no significant change observed in expression of other genes HRI (p=.386), GADD34 (p=.141), CHOP (p=.077). Our study shows that the induction of CHOP (pro apoptotic transcription factor) which strongly depends on the expression of ATF4 induces apoptosis rather than adaptive gene expression in erythroid precursors of HbE-β thalassemia.Table 1Expression of stress related genes in erythroid cells of controls and HbE-β thalassemia patientsHRIGADD34CHOPATF4Median(range)Median(range)Median(range)Median(range)ReticulocyteCONTROL1.070.190.51.32(0.05-4.39)(0.06-1.04)(0.14-1)(0.05-3.72)HbE-β1.70.590.875.87(0.10-12)(0.04-3.8)(0.12-7.77)(2.5-387.49)P-value0.350.0880.0460.000Day 15CONTROL4.221.292.771.43(2 -10.88)(1-3.95)(0.82-6.57)(0.53-3.9)HbE-β11.7913.8627.893.36(2.38-55)(2.84-128)(1.96-34.79)(1.14-8.42)P-value0.0590.00870.0240.028 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Growth and Antioxidant-Related Effects of the Reestablished Ascorbic Acid Pathway in Zebrafish (Danio rerio) by Genomic Integration of L-Gulonolactone Oxidase From Cloudy Catshark (Scyliorhinus torazame).

Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.