Fluorescent PCR Research Articles

Development of a Quantitative Multiplex PCR to Detect Three Common Alpha Thalassemia Deletions

Alpha thalassemia is an autosomal recessive genetic disorder with a high prevalence in the Middle East. The severe form of alpha-thalassemia is incompatible with life and can cause significant obstetric complications in the mother. Therefore, it is important to determine the genotype in parents who have a chance of having a fetus with one of the severe forms of this disease. A total of 112 samples that were previously analyzed for common alpha thalassemia mutations in Iran were used in this study. A new multiplex PCR including quantitative polymerase chain reaction to amplify the homologous regions of the alpha-globin gene cluster and fluorescent gap PCR was designed to identify −α3.7, −α4.2, --MED deletions. The ROC curve was used to determine the optimum cutoff points. Statistical analysis showed that there is a significant difference between the peak height ratios for different genotypes. The peak corresponding to the 297 bp fragment resulting from the amplification of the allele with MED-I deletion was detected in all the samples with this deletion. Different cutoffs for a range of sensitivities and specificities were determined by the ROC curve. The suggested method can identify three common large deletions in the alpha-globin gene cluster. A study with a larger sample size can provide more accurate information about the sensitivity and specificity of this test.

Infecção mista de Bartonella e Eperythrozoon em um cão - relato de caso

ABSTRACT A March male Golden, weighing 7.6kg, presented with gradual weight loss, high body temperature, depression, poor appetite and thirst, and vomiting before consultation. The results showed that the erythrocytes, hematocrit, hemoglobin, and platelets were lower than the reference values. The diagnosis of mixed infection with haematocrit and eosinophilic bodies was confirmed by real-time fluorescence PCR of whole blood, which was positive for haematocrit and eosinophilic bodies. The dog was treated with doxycycline and ceftriaxone, and the dog fully recovered after 2 weeks with blood transfusion, symptomatic treatment, and supportive therapy. This indicates that the disease can be treated well by a comprehensive treatment approach.

Prognostic significance of expression of myogenin and OCT4 in glioma.

Glioma is the most common extracranial solid tumor of children. The malignant (cancer) cells form in neural crest cell of the adrenal gland. In this study we investigate the expressions of myogenin and OCT4 in Glioma which might be used as prognostic makers for this deteriorated disease. We used Quantitative Fluorescence PCR (QF-PCR), Enzyme-linked immunosorbent assay (ELISA), and Western-blotting to measure the expression level of myogenin and OCT4 in surgical removed glioma from 41 patients in our hospital. Compared to the healthy children the expression of myogenin and OCT4 was significantly increased in both mRNA and protein level in Glioma tumor cells. In addition, these expressions increased as glioma deterioration. These findings suggesting the expression of myogenin and Oct4 may be useful indicators for predicting the prognosis of children with glioma.

Hesperetin attenuates cognitive dysfunction via SIRT6/NLRP3 pathway in scopolamine-induced mice.

Neuroinflammation is a critical feature in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). Hesperetin can exert anti-inflammatory, antioxidant and other neuroprotective effects. In this study, the scopolamine (SCOP)-induced cognitive dysfunction in mice model was used to evaluate the neuroprotective effects of hesperetin. Behavioral tests (Morris water maze, open field, and novel object recognition tests) were conducted to evaluate the effect of hesperetin on cognitive dysfunction behaviors. Nissl staining and Immunofluorescence were used to evaluate hippocampal neuronal damage and microglial activation in mice. The levels of proinflammatory factors, oxidant stress, and the cholinergic neurotransmitter were detected by real-time quantitative fluorescence PCR (RT-qPCR) or biochemical reagent kits. Western blotting was used to detect the relative protein expression of the sirtuin 6 (SIRT6) / NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway. Results showed that hesperetin could ameliorate SCOP-induced cognitive impairment and neuronal damage, and regulate the levels of cholinergic neurotransmitters in the hippocampal of AD mice. Hesperetin could also enhance antioxidant defense by regulating the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). Hesperetin exerted anti-neuroinflammation effects through inhibiting of microglia activation and down-regulating the mRNA transcript levels of inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Meanwhile, hesperetin could attenuate the expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), thioredoxin-interacting protein (TXNIP), and caspase-1 p20 and upregulate the expression of SIRT6 in SCOP-induced mice. Overall, our study suggested that hesperetin might ameliorate SCOP-induced cognitive dysfunction by improving cholinergic system dysfunction and suppressing oxidative stress and attenuating neuroinflammation via SIRT6/NLRP3 pathway in mice.

An Optimized Environmental DNA Method to Improve Detectability of the Endangered Sichuan Taimen (Hucho bleekeri)

Environmental DNA (eDNA) techniques have emerged as a cost-effective and non-invasive strategy for monitoring the distribution of endangered aquatic species. Despite their numerous advantages, operational uncertainty at each step of the process represents a significant challenge that can impact the reliability of results. Hence, the optimization of the eDNA analytic protocol is of utmost importance. In this study, the rare and endangered fish species Sichuan taimen was chosen as the target species to evaluate the concordance between different approaches (tissue, pond, and field water samples) for eDNA detection. The results showed that membrane filtration, collection of a 2-L water sample, and utilization of the DNeasy Tissue and Blood DNA extraction kit were suitable for the analysis of DNA in water samples. Furthermore, the mtDNA D-loop region demonstrated potential as a specific molecular marker for the precise identification of Sichuan taimen. Our results indicated that TaqMan fluorescence PCR can serve as the optimal detection method for reliable identification of target DNA even at extremely low concentrations in the field. This study established an optimized eDNA analysis protocol for Sichuan taimen detection, which will provide the basis for future resource investigations aimed at protection of this endangered aquatic species.

"Huayu Tongluo" moxibustion improves learning-memory ability and promotes myelin regeneration by regulating Sonic Hedgehog signaling pathway in vascular dementia rats

To investigate the effect of "Huayu Tongluo" (resolving blood stagnation to dredge meridian-collaterals) moxibustion on remyelination and Sonic Hedgehog (Shh) signaling pathway in the corpus callosum of vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. Male Wistar rats were randomized into sham-operation, model, medication and moxibustion groups, with 12 rats in each group.The VD model was established by bilateral common carotid artery occlusion. Moxibustion was applied to "Shenting"(GV24), "Baihui"(GV20) and "Dazhui"(GV14) for 20 min once a day, 7 d as a treatment course, for 3 courses, with one day's rest between every two courses. Rats of the medication group were treated by gavage of 10 mg/kg of chloromastine solution once a day, and the course of treatment was the same as that of the moxibustion group. The rat's learning-memory ability was assessed by Morris water maze test (escape latency). The neurological deficits were evaluated by using Longa's scale.The mRNA and protein expressions of Shh and Gli1 in the corpus callosum were measured by quantitative real-time fluorescence PCR and Western blot, separately. The ultrastructure of the myelin sheath and myelinated axons was observed under transmission electron microscopy (TCM). Compared with the sham-operation group, the neurologic score and escape latency were significantly increased and prolonged (P<0.01), and the mRNA and protein expression levels of Shh and Gli1 and the number of myelinated axons were obviously decreased in the model group (P<0.01). In comparison with the model group, the escape latency was apparently shortened (P<0.05), while the mRNA and protein expression levels of Shh and Gli1 as well as the number of myelinated axons were strikingly increased in both moxibustion and medication groups (P<0.01). Results of TCM showed that in the model group, the arrangement of myelin coil structures was sparse and fuzzy, and some structures were bulged and disbanded. The oligodendrocytes were irregular, and the number of myelin sheath was rare. These situations were relatively milder in both moxibustion and medication groups. "Huayu Tongluo" moxibustion can promote the differentiation and maturation of oligodendrocyte precursor cells after cerebral ischemia by regulating the expressions of Shh and Gli1 in Shh signaling pathway, thus promoting the regeneration of cerebral white matter myelin sheaths in VD rats, which may contribute to improving learning-memory ability.

High Wnt2 Expression Confers Poor Prognosis in Colorectal Cancer, and Represents a Novel Therapeutic Target in BRAF-Mutated Colorectal Cancer.

Background and Objectives: We aimed to investigate the role of Wnt2 expression in colorectal cancer (CRC) prognosis and evaluate its potential as a therapeutic target in BRAF-mutated CRC. Materials and Methods: Exactly 136 samples of formalin-fixed paraffin-embedded CRC tissue specimens were obtained from patients who underwent surgical resection for CRC. The gene mutation status of the samples was detected using fluorescence PCR. Wnt2 expression was detected using immunohistochemistry. Survival curves with high Wnt2 expression and BRAF mutations were compared using the Kaplan-Meier method. A nomogram was constructed to determine the estimated overall survival probability. We also predicted the 3-year and 5-year survival rates for patients with high Wnt2 expression and BRAF mutations. In total, 50 samples of BRAF-mutated CRC were collected and detected Wnt2 expression by immunohistochemistry. The Chi-squared test was used to analyze the association between Wnt2 expression and BRAF-mutated CRC. Results: High Wnt2 expression and BRAF mutations are associated with poor prognosis of CRC. Multivariate survival analyses indicated that high Wnt2 expression and BRAF mutations are significant independent predictors of CRC prognosis. Furthermore, high Wnt2 expression was significantly associated with BRAF-mutated CRC, and Wnt2 may be a potential therapeutic target for BRAF-mutated CRC. Conclusions: High Wnt2 expression confers poor prognosis in colorectal cancer and represents a novel therapeutic target in BRAF-mutated CRC.

Prenatal diagnosis and genetic analysis of a fetus with partial deletion of Yq and mosaicism of 45,X

To carry out prenatal diagnosis and genetic analysis for a fetus with disorders of sex development (DSDs). A fetus with DSDs who was identified at the Shenzhen People's Hospital in September 2021 was selected as the study subject. Combined molecular genetic techniques including quantitative fluorescence PCR (QF-PCR), multiplex ligation-dependent probe amplification (MLPA), chromosomal microarray analysis (CMA), quantitative real-time PCR (qPCR), as well as cytogenetic techniques such as karyotyping analysis and fluorescence in situ hybridization (FISH) were applied. Ultrasonography was used to observe the phenotype of sex development. Molecular genetic testing suggested that the fetus had mosaicism of Yq11.222qter deletion and X monosomy. Combined with the result of cytogenetic testing, its karyotype was determined as mos 45,X[34]/46,X,del(Y)(q11.222)[61]/47,X,del(Y)(q11.222),del(Y)(q11.222)[5]. Ultrasound examination suggested hypospadia, which was confirmed after elective abortion. Combined the results of genetic testing and phenotypic analysis, the fetus was ultimately diagnosed with DSDs. This study has applied a variety of genetic techniques and ultrasonography to diagnose a fetus with DSDs with a complex karyotype.

A novel ADGRG2 truncating variant associated with X-linked obstructive azoospermia in a large Chinese pedigree.

In this study, we aimed to identify sterility-related variants in a Chinese pedigree with male infertility and to reveal the different phenotypes and intracytoplasmic sperm injection (ICSI) outcomes of the affected members. Physical examinations were performed on male patients. G-band karyotype analysis, copy number variation sequencing, and quantitative fluorescent PCR were conducted to detect common chromosomal disorders in the probands. Whole-exome sequencing and Sanger sequencing were applied to identify the pathogenic genes and the protein expression changes caused by the very mutation were identified by Western Blot in vitro. A novel nonsense mutation (c.908C > G: p.S303*) in the ADGRG2 was identified in all infertile male patients of the pedigree, which was inherited from their mothers. This variant was absent from the human genome databases. This mutation was also unexpectedly found in a male member with normal reproductive capability. Members with the mutation had different genitalia phenotypes, ranging from normal to dilated phenotypes of the vas deferens, spermatic veins and epididymis. There was a truncated ADGRG2 protein in vitro after mutation. Of the three patients' wives treated with ICSI, only one successfully gave birth. Our study is the first to report the c.908C > G: p.S303* mutation in the ADGRG2 in an X-linked azoospermia pedigree and is the first to report normal fertility in a member with this mutation, expanding the mutation spectrum and phenotype spectrum of this gene. In our study, ISCI had a success rate of only one-third in couples including men with azoospermia with this mutation.

Abstract 9: Association Between HMOX1 (GT)n Promoter Microsatellite and Prostate Cancer Prognosis

Abstract Purpose: Heme Oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation encoded by HMOX1 gene, has a strong anti-tumoral effect in prostate cancer (PCa). HMOX1 expression levels are modulated by the length of a dinucleotide (GT)n microsatellite polymorphism mapping in its promoter region. PCa is the second most frequent cancer and the sixth leading cause of cancer-related deaths in men worldwide. One of the strongest predictors for newly diagnosed PCa is the International Society of Urological Pathology (ISUP) grade, a histopathology-based classification system, which presents diverse limitations due to tumoral heterogeneity. Consequently, the identification of novel biomarkers is important to optimize PCa prognosis. In this study we genotyped the HMOX1 (GT)n promoter polymorphism and analyze its association with PCa clinico-pathological parameters. Methods: We collected 108 peripheral blood samples from PCa patients from Hospital de Clínicas “Jose de San Martín”, Buenos Aires, Argentina. Genomic DNA was isolated and the (GT)n was genotyped by fluorescent PCR and capillary electrophoresis. Randomly selected samples were cloned and sequenced by Sanger to determine the length of the microsatellite and validate the results. We considered a statistically significant difference between groups if p≤0.05. Results: The length of the (GT)n ranged from 11 to 40 repeats. HMOX1 alleles were dichotomized as “Long (l)” (≥30 repeats) or “Short (s)” (&amp;lt;30 repeats) based on the median length; and genotypes were categorized as homozygous “HMOX1ss, homozygous “HMOX1ll”, or heterozygous “HMOX1sl”. We found an association between the “HMOX1ss” genotype and higher serum prostate specific antigen (sPSA) levels among patients with &amp;lt;10 ng/mL sPSA (p&amp;lt;0.01). Furthermore, the proportion of “HMOX1ss” patients increased with the ISUP grade, whereas “HMOX1ll” or “HMOX1sl” genotypes were more frequent in lower ISUP grades (ptrend&amp;lt;0.05). Finally, “HMOX1ss” patients had 4-fold higher risk of biochemical relapse (HR=4.03, p&amp;lt;0.05). Conclusion: “HMOX1ss” patients present more aggressive phenotypes compared with the other genotypes. HMOX1 polymorphism has the potential to classify patients in different groups to predict the severity of PCa tumors, thus evidencing potential to support clinicians on the decision making and improving clinical management of PCa at an early stage through a non-invasive sample. Citation Format: Gaston Pascual, Agustina Sabater, Rocio Seniuk, Ines Achinelli, Pamela Freiria, Carlos Scorticati, Osvaldo Mazza, Ayelen Toro, Elba Vazquez, Geraldine Gueron, Javier Cotignola. Association Between HMOX1 (GT)n Promoter Microsatellite and Prostate Cancer Prognosis [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 9.

BRCA1 and RAD51 methylation impact on outcome in patients with advanced ovarian cancer: A PAOLA-1 ancillary study.

5559 Background: The PAOLA-1/ENGOT-ov25 trial showed that adding olaparib (ola) to maintenance bevacizumab (bev) after first-line therapy led to a progression-free and overall survival benefit in advanced ovarian high-grade carcinoma (AOC) patients with homologous recombination deficiency (HRD) (Myriad MyChoiceR Genomic Instability Score) or BRCA1/2 mutations (BRCAmut). Here, we address the impact of BRCA1 and RAD51C epimutations to improve our understanding of GIS beyond BRCAmut. Methods: BRCA1 and RAD51C methylation analysis was assessed and quantified in pre-treatment biopsies (66%), after neoadjuvant chemotherapy (NACT) (20%) or unknown (14%) from bisulfite converted DNAs using fluorescent methylation specific PCR and methylation specific droplet digital PCR (ddPCR). Methylation status was correlated to clinical data, BRCA1/2 mutations, GIS and HRD scores, PFS (PFS1, PFS2) and OS. R (r-project.org) was used for statistical analysis. Results: Among the 537 patients randomized to maintenance ola + bev and 269 to placebo (pbo) + bev, 348 and 171 were available for methylation analysis. Their baseline molecular and clinical characteristics were well balanced with those of the entire cohort. Promoter methylation was identified in 67 (12.9%) samples for BRCA1 and 25 (4.8%) for RAD51C (4 were methylated on both genes). Methylation and BRCAmut were mutually exclusive except for 3 samples. Mean GIS scores were 62.5 [59.6-65.5]; 59.4 [57.2-61.5]; 54.2 [50.5-57.8]; 23.4 [21.6-25.2] for BRCA1 or RAD51C methylated ( met-tumors), BRCAmut, non-mut/non-met HRD+ ( b-/m-HRD+) and HRP (proficient) tumors respectively. Among met-tumors 92% (66/72) were GIS positive (&gt;42). The mean GIS score of Met-tumors were significantly higher than that of b-/m- HRD+ samples (p=0,009). Benefit of adding ola maintenance to bev was in a similar between patients with met AOC and those with b-/m- HRD+ tumors ( table). Conclusions: Methylated BRCA1/RAD51 tumors are HRD+ and provide to ovarian cancer patients a similar clinical benefit of ola+bev as patients with b-/m- HRD+ tumors. Methylation assessment represents a rapid and cost effective tool; which coupled with BRCA1-2 somatic testing allows the identification of the majority (81%) of HRD+ AOC. Clinical trial information: NCT02477644 . [Table: see text]

Lapachol treats non-alcoholic fatty liver disease by modulating the M1 polarization of Kupffer cells via PKM2

AimThis study investigated the mechanism of action of lapachol (LAP) against non-alcoholic fatty liver disease (NAFLD). MethodsPrimary Kupffer cells (KCs) of rats were used for in-vitro experiments. The proportion of M1 cells was assayed by flow cytometry, the levels of M1 inflammatory markers were determined by enzyme-linked immunosorbent assay (ELISA) combined with real-time quantitative fluorescence PCR (RT-qPCR), the expression of p-PKM2 was detected by Western-Blotting. A SD rat model of NAFLD was established with high-fat diet. Following LAP intervention, the changes in blood glucose/lipid, insulin resistance and liver function were detected, and the hepatic histopathologic changes were examined by histological staining. ResultsThe results showed that LAP could inhibit the M1 polarization of KCs, lower the levels of inflammatory cytokines, and suppress the activation of PKM2. The effect of LAP could be counteracted after using PKM2 inhibitor PKM2-IN-1 or knocking out PKM2. Small molecule docking revealed that LAP could inhibit the phosphorylation process of PKM2 by binding to ARG-246, the phosphorylation site of PKM2. In rat experiments, LAP could ameliorate the liver function and lipid metabolism of NAFLD rats, and inhibit the hepatic histopathologic changes. ConclusionOur study found that LAP can inhibit the phosphorylation of PKM2 by binding to PKM2-ARG-246, thereby regulating the M1 polarization of KCs and inhibiting the inflammatory response of liver tissues to treat NAFLD. LAP has potential as a novel pharmaceutical for treating NAFLD.

Characterization of wheat Wrab18 gene promoter and expression analysis under abiotic stress.

Promoters play key roles in plant gene expression in complex and varied natural environments. The type and amount of cis-acting elements in the promoter sequence tend to indicate the response of genes to induction factors. WRAB18 is a group III member of the late embryogenesis abundant (LEA) protein family that performs multiple functions in plant stress physiology. To elucidate the particularly biological effects of WRAB18 on stress, exploration of its promoter sequence is necessary. In this study, the full-length and promoter sequences of Wrab18 were isolated from the Zhengyin 1 cultivar of Triticum aestivum. The gene sequences and cis-acting elements in the promoter were analyzed using the Plant Promoter Database and bioinformatics methods. The results showed that Wrab18 possessed one intron with 100bp, the promoter sequence contained various stress-related cis-acting elements, and the functionality of the promoter was checked using green fluorescent protein (GFP) marker protein expression by transient assay in Nicotiana benthamiana. Furthermore, based on promoter prediction analysis, quantitative real-time fluorescent PCR results confirmed the response of gene expression levels to stress factors. In summary, the promoter sequence of Wrab18 plays a role in plant stress responses, contains multiple cis-acting elements, and provides insights into the role of WRAB18 in plant resilience to stress. This study has guiding significance for further studies of gene function and mechanism of action, and lays a theoretical foundation for improving wheat quality.

Epidemiological characteristics of New Delhi Metallo-β-Lactamase-producing Enterobacteriaceae in the Fourth hospital of Hebei Medical University

The epidemiological characteristics of New Delhi Metallo-β-Lactamase-Producing (NDM) Enterobacteriaceae were analyzed to provide theoretical support for clarifying the distribution characteristics of carbapenem-resistant Enterobacteriaceae (CRE) in the hospital environment and early identification of susceptible patients. From January 2017 to December 2021,42 strains of NDM-producing Enterobacteriaceae were gathered from the Fourth Hospital of Hebei Medical University, primarily Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. The micro broth dilution method combined with the Kirby-Bauer method was used to determine the minimal inhibitory concentrations (MICs) of antibiotics. The carbapenem phenotype was detected by the modified carbapenem inactivation method (mCIM) and EDTA carbapenem inactivation method (eCIM). Carbapenem genotypes were detected by colloidal gold immunochromatography and real-time fluorescence PCR. The results of antimicrobial susceptibility testing showed that all NDM-producing Enterobacteriaceae were multiple antibiotic resistant, but the sensitivity rate to amikacin was high. Invasive surgery prior to culture, the use of excessive amounts of different antibiotics, the use of glucocorticoids, and ICU hospitalization were clinical characteristics of NDM-producing Enterobacteriaceae infection. Molecular typing of NDM-producing Escherichia coli and Klebsiella pneumoniae was carried out by Multilocus Sequence Typing (MLST), and the phylogenetic trees were constructed. Eight sequence types (STs) and two NDM variants were detected in 11 strains of Klebsiella pneumoniae, primarily ST17, and NDM-1. A total of 8 STs and 4 NDM variants were detected in 16 strains of Escherichia coli, mainly ST410, ST167, and NDM-5. For high-risk patients who have CRE infection, CRE screening should be done as soon as feasible to adopt prompt and efficient intervention measures to prevent outbreaks in the hospital.

Initial development and testing of an exhaled microRNA detection strategy for lung cancer case–control discrimination

For detecting field carcinogenesis non-invasively, early technical development and case–control testing of exhaled breath condensate microRNAs was performed. In design, human lung tissue microRNA-seq discovery was reconciled with TCGA and published tumor-discriminant microRNAs, yielding a panel of 24 upregulated microRNAs. The airway origin of exhaled microRNAs was topographically “fingerprinted”, using paired EBC, upper and lower airway donor sample sets. A clinic-based case–control study (166 NSCLC cases, 185 controls) was interrogated with the microRNA panel by qualitative RT-PCR. Data were analyzed by logistic regression (LR), and by random-forest (RF) models. Feasibility testing of exhaled microRNA detection, including optimized whole EBC extraction, and RT and qualitative PCR method evaluation, was performed. For sensitivity in this low template setting, intercalating dye-based URT-PCR was superior to fluorescent probe-based PCR (TaqMan). In application, adjusted logistic regression models identified exhaled miR-21, 33b, 212 as overall case–control discriminant. RF analysis of combined clinical + microRNA models showed modest added discrimination capacity (1.1–2.5%) beyond clinical models alone: all subjects 1.1% (p = 8.7e−04)); former smokers 2.5% (p = 3.6e−05); early stage 1.2% (p = 9.0e−03), yielding combined ROC AUC ranging from 0.74 to 0.83. We conclude that exhaled microRNAs are qualitatively measureable, reflect in part lower airway signatures; and when further refined/quantitated, can potentially help to improve lung cancer risk assessment.

Effect and mechanism of imbalance via Th9 cells and Th17/Treg cells in inflammatory and fibrotic phases of pulmonary fibrosis in mice

ABSTRACT We investigate the role and mechanism of imbalance via Th9 cells and Th17/Treg cells in the inflammatory and fibrotic phases of pulmonary fibrosis in mice. A total of mice were split into normal saline (control group) and inflammation and fibrosis mouse models (study group) randomly, and lung tissues and bronchoalveolar lavage fluid (BALF) were obtained from mice at the inflammatory and fibrotic phases on the 7th and 28th day, respectively. The degenerative changes in the mouse lung tissue were then visible using H&E staining. The expression of CCR6 and IL-9 in the lung tissues of two groups was examined through an immunohistochemistry assay. Fluorescence PCR was used to assess the expression of PU.1 mRNA in BALF, and flow cytometry was performed to identify the expression of Th17 and Treg. (1). The level of pulmonary fibrosis and lung inflammation in the research group was significantly higher than in the control group. (2). The expression of Th17, CCR6, IL-9 and PU.1 mRNA was substantially higher (P<0.05) in the research group at different time points; the expression level of Treg cells was considerably lower (P<0.05) in the research group than in the control group. (3). CCR6, IL-9 and PU.1 mRNA levels were statistically directly associated (P<0.05) with Th17 and inversely correlated 40 with Regulatory T cells (Tregs). CCR6 and Th9 cells may be involved in 45 developing Th17/Treg imbalance in the immune inflammation of pulmonary fibrosis, which promotes fibrocyte proliferation in lung tissue.

Identification of the Function of the Pathogenesis-Related Protein GmPR1L in the Resistance of Soybean to Cercospora sojina Hara.

Pathogenesis-related proteins, often used as molecular markers of disease resistance in plants, can enable plants to obtain systemic resistance. In this study, a gene encoding a pathogenesis-related protein was identified via RNA-seq sequencing analysis performed at different stages of soybean seedling development. Because the gene sequence showed the highest similarity with PR1L sequence in soybean, the gene was named GmPR1-9-like (GmPR1L). GmPR1L was either overexpressed or silenced in soybean seedlings through Agrobacterium-mediated transformation to examine the resistance of soybean to infection caused by Cercospora sojina Hara. The results revealed that GmPR1L-overexpressing soybean plants had a smaller lesion area and improved resistance to C. sojina infection, whereas GmPR1L-silenced plants had low resistance to C. sojina infection. Fluorescent real-time PCR indicated that overexpression of GmPR1L induced the expression of genes such as WRKY, PR9, and PR14, which are more likely to be co-expressed during C. sojina infection. Furthermore, the activities of SOD, POD, CAT, and PAL were significantly increased in GmPR1L-overexpressing soybean plants after seven days of infection. The resistance of the GmPR1L-overexpressing lines OEA1 and OEA2 to C. sojina infection was significantly increased from a neutral level in wild-type plants to a moderate level. These findings predominantly reveal the positive role of GmPR1L in inducing resistance to C. sojina infection in soybean, which may facilitate the production of improved disease-resistant soybean cultivars in the future.

Variable fragment length allele-specific polymerase chain reaction (VFLASP), a method for simple and reliable genotyping

Single-nucleotide polymorphism (SNP) is a substitution of a single nucleotide at a specific position in the genome. Until now, 585 million SNPs have been identified in the human genome, and therefore, a widely applicable method is desirable to detect a specific SNP. Herein we report a simple and reliable genotyping assay, which seems to be suitable for medium and small size laboratories, as well, to easily genotype most of the SNPs. In our study, all of the possible base variations (A-T, A-G, A-C, T-G, T-C, G-C) were tested to prove the general feasibility of our technique. The basis of the assay is a fluorescent PCR, in which both allele-specific primers, differing only at the 3′ end according to the sequence of the SNP, were present, and the length of one of them was modified with 3 bp by adding an adapter sequence to the 5’ end of that primer. The competitive presence of both allele-specific primers excludes the false amplification of the absent allele (which can happen in simple allele-specific PCR (AS-PCR)) and ensures the amplification of the proper allele(s). Unlike other complicated genotyping methods that use of manipulation of fluorescent dyes for genotyping, we apply an approach based on the length of amplicons from different alleles to differentiate between them. In our experiment (named variable fragment length allele-specific polymerase chain reaction (VFLASP)), the investigated six SNPs, containing the six available base variations, gave clear and reliable results after detecting the amplicons by capillary electrophoresis.

Distribution of soil microorganisms in different complex soil layers in Mu Us sandy land.

The soft rock in Mu Us Sandy Land has rich resources and high content of clay minerals. The combination of soft rock with sand can play a certain role in sand fixation and promote the green development of ecological environment. In this paper, the aeolian sandy soil in Mu Us Sandy was taken as the research object, and it was mixed with soft rock to form composite soil. The four volume ratios of soft rock to sand were respectively 0:1, 1:5, 1:2 and 1:1. And CK, P1, P2 and P3 were used to represent the above four volume ratios in turn. By means of quantitative fluorescent PCR and high throughput sequencing, 16S rRNA gene abundance and community structure were investigated. The results showed that the soil organic carbon (SOC) and total nitrogen (TN) contents in 0-30cm soil layer were higher. Compared with CK, the SOC of P2 was improved by 112.77% and that of P1 was 88.67%. The content of available phosphorus (AP) and available potassium (AK) was higher in 30-60cm soil layer, and P3 was more effective. The abundance of 16S rRNA gene in the mixed soil bacteria ranged from 0.03×109 to 0.21×109 copies g-1 dry soil, which was consistent with the changes of nutrients. Under different soil layers, the three dominant bacteria in the mixed soil were the same, namely Phylum Actinobacteriota, Phylum Proteobacteria and Phylum Chloroflexi, and there were more unique genera in each soil layer. Both bacteria ɑ and β diversity showed that the community structure of P1 and P3 in 0-30cm soil layers was similar, and that of P1 and P2 in 30-60cm soil layers was similar. AK, SOC, AN (ammonium nitrogen), TN and NN (nitrate nitrogen) were the main factors contributing to the differentiation of microbial community structure under different compound ratios and soil layers, and Phylum Actinobacteria has the largest correlation with nutrients. The results showed that the soft rock could improve the quality of sandy soil, and that the growth of microbial growth was dependent on the soil physicochemical characteristics. The results of this study will be helpful to the study of the microscopical theory for the control of the wind-blown sand and the ecology of the desert.

Effect of hemX gene deletion on heme synthesis in Bacillus amyloliquefaciens

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.