Drug resistance characteristics and molecular typing of Escherichia coli isolates from neonates in class A tertiary hospitals: A multicentre study across China.

OBJECTIVES: Escherichia coli is a common pathogen causing invasive bacterial infections in neonates. In recent years, clinical antimicrobial susceptibility testing has demonstrated an increased rate of drug-resistant E. coli infections. This study aimed to analyse the resistance characteristics of E. coli against common antimicrobial agents, and perform multilocus sequence typing (MLST) in clinical strains of E. coli collected from Chinese neonates. METHODS: Culture-positive specimens of E. coli were collected from neonates in seven class A tertiary hospitals located in seven cities across different provinces in China between November 2019 and October 2020. E. coli isolated from these specimens were subjected to antimicrobial susceptibility testing (by broth microdilution method), extended-spectrum ß-lactamase (ESBL) detection, and MLST. RESULTS: A total of 223 E. coli strains were isolated, with an overall resistance rate of 87.4%, an ESBL-positive rate of 48.0%, and a multidrug resistance rate of 42.2%. Among the 20 antimicrobial agents tested, E. coli strains showed the highest resistance rates against cefotaxime (59.2%), trimethoprim/sulfamethoxazole (56.5%), doxycycline (39.9%), ciprofloxacin (36.8%), and aztreonam (31.0%). The resistance rates of E. coli strains isolated from children's hospitals against piperacillin/tazobactam, cefotaxime, ciprofloxacin, trimethoprim/sulfamethoxazole, and carbapenems, were significantly higher than those of strains isolated from maternity and child health hospitals. The primary E. coli multilocus sequence types were ST1193, ST95, ST73, ST410, and ST131. The ESBL production rates and multidrug resistance rates of ST1193, ST410, and ST131 were significantly higher than those of ST95 and ST73. Significantly, more strains of E. coli ST1193 and ST410 were isolated from children's hospitals than from maternity and child health hospitals. CONCLUSIONS: The rates of antimicrobial agent resistance in E. coli isolates from hospitalised neonates in China were high. The increased number of strains of E. coli ST1193 and ST410 was the reason for higher resistance rates to multiple antimicrobial agents in E. coli from children's hospitals compared with those from maternal and child health hospitals.

Vitamin D Ameliorates Apoptosis and Inflammation by Targeting the Mitochondrial and MEK1/2-ERK1/2 Pathways in Hyperoxia-Induced Bronchopulmonary Dysplasia.

Purpose: Bronchopulmonary dysplasia (BPD) is a common and severe complication in preterm infants. Vitamin D (VitD) has been reported to protect against BPD; however, its role in the mitochondria-mediated and MEK1/2-ERK1/2 pathways has not yet been reported. Methods: We first performed in vivo studies using neonatal C57BL/6 mice in which we induced BPD by exposing them to a hyperoxic environment (85% O2). The mice were divided into room air (RA; 21% O2), RA+VitD, BPD, and BPD+VitD groups. Hematoxylin and eosin and Masson's trichrome staining were used to evaluate lung injury. Inflammation and apoptosis were measured using ELISA, RT-qPCR, and TUNEL assays. We then analyzed BEAS-2B cells divided into the same groups along with an additional BPD+VitD+inhibitor group. Mitochondrial apoptosis was evaluated by transmission electron microscopy, mitochondrial membrane potential, and Western blotting. We then used VDR-shRNA to silence the Vitamin D Receptor (VDR) in the BEAS-2B cells. The inflammation, apoptotic rate, and the phosphorylated forms of MEK1/2 and ERK1/2 in cells were detected by RT-qPCR, flow cytometry, and Western blotting. Results: The mean linear intercept, septal thickness, and abnormal fibrosis increased, while radial alveolar count decreased in BPD lungs compared to RA lungs. VitD administration was able to ameliorate the phenotype in BPD lungs. IL-6, IFN-γ, and TNF-α expression and the apoptotic rate decreased in the BPD+VitD lung group. VitD pretreatment restored abnormal mitochondrial morphology, reduced mitochondrial membrane loss, and reduced the expression of cleaved caspase-3, Bax, and Bcl-2 in BEAS-2B cells. VitD administration also reduced IL-6, IFN-γ, and TNF-α mRNA, as well as pMEK1/2 and pERK1/2 expression and apoptosis rate in cells exposed to hyperoxia. Conclusion: We concluded that VitD treatment ameliorated apoptosis and inflammation by targeting the mitochondrial pathway and via the MEK1/2-ERK1/2 signaling pathway in BPD, thus supporting its potential therapeutic use in this condition.

Serum Vitamin D Insufficiency in Hospitalized Full-Term Neonates at a Tertiary Hospital in Eastern China.

Objective: This study explored the status of serum vitamin D in hospitalized full-term neonates at a tertiary hospital in eastern China. Methods: A prospective study was conducted among 471 hospitalized full-term neonates at the Children's Hospital of Soochow University between January 1 and June 20, 2020. Perinatal clinical data, serum 25-hydroxyvitamin D (25(OH)D3), laboratory examinations, serum calcium levels, and immune function were obtained and analyzed. We explored and analyzed the risk factors for vitamin D insufficiency or deficiency, and we also attempted to determine correlations between vitamin D and its influence on immunity. Results: The mean serum 25(OH)D3 was 33.65±6.07ng/ml.The prevalence of vitamin D insufficiency was 28.24%,vitamin D sufficiency was 71.76%, there was no vitamin D deficiency. The serum 25-(OH)D3 in singleton neonate was higher than twins or multiple infants (t = -10.918, P = 0.000). The serum 25-(OH)D3 were higher in neonates who born in spring and summer than in winter (H = 13.443, P = 0.001). The serum 25-(OH)D3 in appropriate for gestational age (AGA) and large for gestational age (LGA) neonates were higher than small for gestational age (SGA) (H = 7.686, P = 0.021). The serum 25-(OH)D3 were higher in neonates whose mothers had no underlying diseases than those with certain immunological and infectious diseases (F = 12.417, P = 0.000). The serum 25-(OH)D3 in neonates whose mothers had none or one perinatal complication were higher than those with two or more (F = 13.299, P = 0.000). The neonates with eosinophils ≤5% or normal platelet counts or serum Ca++ ≥0.9 mmol/L have higher serum 25-(OH)D3. Neonates born in winter were at risk for vitamin D insufficiency, and the incidence of infectious pneumonia, sepsis, cytomegalovirus infection, and hypocalcemia in the vitamin D insufficiency group were higher than sufficiency group (P < 0.05). The serum CD3+, CD3+CD4+, and IgA levels in vitamin D sufficiency neonates were significantly higher than those in insufficiency group (P < 0.05). Conclusion: The prevalence of vitamin D insufficiency was 28.24%, and birth in winter was a risk factor for vitamin D insufficiency in hospitalized full-term neonates in Suzhou area. Neonates with infectious pneumonia, sepsis, cytomegalovirus infection, and hypocalcemia exhibited a high risk of vitamin D insufficiency. The serum CD3+, CD3+CD4+, and IgA levels in neonates with vitamin D insufficiency were lower.

Bacterial Community Assembly, Succession, and Metabolic Function during Outdoor Cultivation of Microchloropsis salina.

Outdoor cultivation of microalgae has promising potential for renewable bioenergy, but there is a knowledge gap on the structure and function of the algal microbiome that coinhabits these ecosystems. Here, we describe the assembly mechanisms, taxonomic structure, and metabolic potential of bacteria associated with Microchloropsis salina cultivated outdoors. Open mesocosms were inoculated with algal cultures that were either free of bacteria or coincubated with one of two different strains of alga-associated bacteria and were sampled across five time points taken over multiple harvesting rounds of a 40-day experiment. Using quantitative analyses of metagenome-assembled genomes (MAGs), we tracked bacterial community compositional abundance and taxon-specific functional capacity involved in algal-bacterial interactions. One of the inoculated bacteria (Alteromonas sp.) persisted and dispersed across mesocosms, whereas the other inoculated strain (Phaeobacter gallaeciensis) disappeared by day 17 while a taxonomically similar but functionally distinct Phaeobacter strain became established. The inoculated strains were less abundant than 6 numerically dominant newly recruited taxa with functional capacities for mutualistic or saprophytic lifestyles, suggesting a generalist approach to persistence. This includes a highly abundant unclassified Rhodobacteraceae species that fluctuated between 25% and 77% of the total community. Overall, we did not find evidence for priority effects exerted by the distinct inoculum conditions; all mesocosms converged with similar microbial community compositions by the end of the experiment. Instead, we infer that the 15 total populations were retained due to host selection, as they showed high metabolic potential for algal-bacterial interactions such as recycling alga-produced carbon and nitrogen and production of vitamins and secondary metabolites associated with algal growth and senescence, including B vitamins, tropodithietic acid, and roseobacticides. IMPORTANCE Bacteria proliferate in nutrient-rich aquatic environments, including engineered algal biofuel systems, where they remineralize photosynthates, exchange secondary metabolites with algae, and can influence system output of biomass or oil. Despite this, knowledge on the microbial ecology of algal cultivation systems is lacking, and the subject is worthy of investigation. Here, we used metagenomics to characterize the metabolic capacities of the predominant bacteria associated with the biofuel-relevant microalga Microchloropsis salina and to predict testable metabolic interactions between algae and manipulated communities of bacteria. We identified a previously undescribed and uncultivated organism that dominated the community. Collectively, the microbial community may interact with the alga in cultivation via exchange of secondary metabolites which could affect algal success, which we demonstrate as a possible outcome from controlled experiments with metabolically analogous isolates. These findings address the scalability of lab-based algal-bacterial interactions through to cultivation systems and more broadly provide a framework for empirical testing of genome-based metabolic predictions.

Predictive values of clinical data,molecular biomarkers, and echocardiographic measurements in preterm infants with bronchopulmonary dysplasia.

Objective: We aimed to use molecular biomarkers and clinical data and echocardiograms that were collected during admission to predict bronchopulmonary dysplasia (BPD) in preterm infants with gestational age ≤32 weeks. Methods: Eighty-two patients (40 with BPD, BPD group and 42 healthy as controls, non-BPD group) admitted to the Department of Neonatology of the Children's Hospital of Soochow University between October 1, 2018, and February 29, 2020, were enrolled in this study at the tertiary hospital. Basic clinical data on the perinatal period, echocardiographic measurements, and molecular biomarkers (N-terminal-pro-B-brain natriuretic peptide, NT-proBNP) were collected. We used multiple logistic regression analysis to establish an early predictive model for detecting BPD development in preterm infants of gestational age ≤32 weeks. We also used a receiver operating characteristic curve to assess the sensitivity and specificity of the model. Results: No significant differences were found between the BPD and non-BPD groups in terms of sex, birth weight, gestational age, incidence of asphyxia, maternal age, gravidity, parity, mode of delivery, premature rupture of membranes >18 h, use of prenatal hormones, placental abruption, gestational diabetes mellitus, amniotic fluid contamination, prenatal infections, and maternal diseases. The use of caffeine, albumin, gamma globulin; ventilation; days of FiO2 ≥ 40%; oxygen inhalation time; red blood cell suspension infusion volume (ml/kg); and proportion of infants who received total enteral nutrition (120 kcal/kg.d) ≥24 d after birth were higher in the BPD group than in the non-BPD group. The levels of hemoglobin, hematocrit, and albumin in the BPD group were significantly lower than those in the non-BPD group. The total calorie intake was significantly lower in the BPD group on the 3rd, 7th, and 14th day after birth than in the non-BPD group (P < 0.05). The incidence rates of patent ductus arteriosus (PDA), pulmonary hypertension, and tricuspid regurgitation were significantly higher in the BPD group than in the non-BPD group (P < 0.05). The serum level of NT-proBNP 24 h after birth was significantly higher in the BPD group than in the non-BPD group (P < 0.05). Serum NT-proBNP levels were significantly higher in infants with severe BPD than in those with mild or moderate BPD (P < 0.05). Conclusion: As there were various risk factors for BPD, a combining clinical data, molecular biomarkers, and echocardiogram measurements can be valuable in predicting the BPD. The tricuspid regurgitation flow rate (m/s), NT-proBNP (pg/ml), ventilator-associated pneumonia, days of FiO2 ≥ 40% (d), red blood cell suspension infusion volume (ml/kg), and proportion of infants who received total enteral nutrition (120 kcal/kg.d) ≥24 d after birth were the most practical factors considered for designing an appropriate model for predicting the risk of BPD.

Prediction of Bronchopulmonary Dysplasia in Preterm Infants Using Postnatal Risk Factors.

Objective: To identify postnatal risk factors for bronchopulmonary dysplasia (BPD) development in preterm infants with gestational age ≤32 weeks. Methods: Seventy-two preterm infants(30 with BPD and 42 non-BPD controls) admitted in the neonatal intensive care unit (NICU) of the Children's Hospital of Soochow University during 2017 were enrolled in this prospective longitudinal study. Perinatal clinical data, a neonatal critical illness score (NCIS), different soluble B7-H3(sB7-H3), and interleukin-18 (IL-18) levels by days after birth were collected. An early predictive model for BPD development was established based on clinical data using multiple logistic regression analysis. And the sensitivity and specificity of the model were assesed by ROC curve. Results: Electrolyte disturbances, hemodynamically significant patent ductus arteriosus (hs-PDA), and the age that infants achieved 120 kcal/kg.d via enteral feeding ≥40 days after birth were found to be associated with the BPD pathogenesis. Serum sB7-H3, IL-18, and NCIS were significantly higher in the BPD group compared to the non-BPD group (p < 0.05). BPD group had significantly lower enteral fluid and caloric intake compared to the non-BPD group at 1, 7, 14, and 28 days after birth. The risk factors were analyzed by multiple logistic regression and a predictive model of a combination of sB7-H3 (day 7), IL-18 (day 14), NCIS, and clinical risk factors was evaluated via ROC curve with an area under the curve (AUC) of 0.960 having sensitivity of 86.7% and a specificity of 97.6%, respectively. Conclusion: The causes of BPD are multifactorial postnatal risk factors. And the combination of sB7-H3 (day 7), IL-18 (day 14), NCIS, and clinical risk factors (electrolyte disturbances, hs-PDA, and the age that infants achieved 120 kcal/kg.d via enteral feeding ≥40 days after birth) might be served as an optimal predictive model for the occurrence of BPD.

Bowtie loaded meander antenna for a high-temperature superconducting terahertz detector and its characterization by the Josephson effect.

In a quasi-optical system, the high temperature superconducting terahertz detector often suffers from a fundamental problem of low coupling efficiency with the terahertz signal, especially for the detector based on YBa2Cu3O7-δ (YBCO) bicrystal Josephson junction (JJ) due to a small normal-state resistance. Here, we developed a bowtie loaded meander antenna to enhance coupling efficiency. Differing from the conventional characterization confining on vector network analyzers, we applied three methods to evaluate the antenna, including the measurements of the maximal size of the first order Shapiro step under per incident power, the coupling efficiency between the antenna and the junction, and voltage responsivity. Furthermore, with simulation analysis, we propose that the inductive reactance of the YBCO bicrystal JJ is around 60 ohms under terahertz irradiation at 210 GHz, thus, the reactance is comparable as that of the antenna.

Changes in the Structure of the Microbial Community Associated with Nannochloropsis salina following Treatments with Antibiotics and Bioactive Compounds.

Open microalgae cultures host a myriad of bacteria, creating a complex system of interacting species that influence algal growth and health. Many algal microbiota studies have been conducted to determine the relative importance of bacterial taxa to algal culture health and physiological states, but these studies have not characterized the interspecies relationships in the microbial communities. We subjected Nanochroloropsis salina cultures to multiple chemical treatments (antibiotics and quorum sensing compounds) and obtained dense time-series data on changes to the microbial community using 16S gene amplicon metagenomic sequencing (21,029,577 reads for 23 samples) to measure microbial taxa-taxa abundance correlations. Short-term treatment with antibiotics resulted in substantially larger shifts in the microbiota structure compared to changes observed following treatment with signaling compounds and glucose. We also calculated operational taxonomic unit (OTU) associations and generated OTU correlation networks to provide an overview of possible bacterial OTU interactions. This analysis identified five major cohesive modules of microbiota with similar co-abundance profiles across different chemical treatments. The Eigengenes of OTU modules were examined for correlation with different external treatment factors. This correlation-based analysis revealed that culture age (time) and treatment types have primary effects on forming network modules and shaping the community structure. Additional network analysis detected Alteromonadeles and Alphaproteobacteria as having the highest centrality, suggesting these species are "keystone" OTUs in the microbial community. Furthermore, we illustrated that the chemical tropodithietic acid, which is secreted by several species in the Alphaproteobacteria taxon, is able to drastically change the structure of the microbiota within 3 h. Taken together, these results provide valuable insights into the structure of the microbiota associated with N. salina cultures and how these structures change in response to chemical perturbations.

Longitudinal Analysis of Microbiota in Microalga Nannochloropsis salina Cultures.

Large-scale open microalgae cultivation has tremendous potential to make a significant contribution to replacing petroleum-based fuels with biofuels. Open algal cultures are unavoidably inhabited with a diversity of microbes that live on, influence, and shape the fate of these ecosystems. However, there is little understanding of the resilience and stability of the microbial communities in engineered semicontinuous algal systems. To evaluate the dynamics and resilience of the microbial communities in microalgae biofuel cultures, we conducted a longitudinal study on open systems to compare the temporal profiles of the microbiota from two multigenerational algal cohorts, which include one seeded with the microbiota from an in-house culture and the other exogenously seeded with a natural-occurring consortia of bacterial species harvested from the Pacific Ocean. From these month-long, semicontinuous open microalga Nannochloropsis salina cultures, we sequenced a time-series of 46 samples, yielding 8804 operational taxonomic units derived from 9,160,076 high-quality partial 16S rRNA sequences. We provide quantitative evidence that clearly illustrates the development of microbial community is associated with microbiota ancestry. In addition, N. salina growth phases were linked with distinct changes in microbial phylotypes. Alteromonadeles dominated the community in the N. salina exponential phase whereas Alphaproteobacteria and Flavobacteriia were more prevalent in the stationary phase. We also demonstrate that the N. salina-associated microbial community in open cultures is diverse, resilient, and dynamic in response to environmental perturbations. This knowledge has general implications for developing and testing design principles of cultivated algal systems.

TdaA regulates Tropodithietic acid synthesis by binding to the tdaC promoter region.

Silicibacter sp. TM1040, a member of the marine Roseobacter clade, produces the antibiotic and quorum signaling molecule tropodithietic acid (TDA), encoded by tdaABCDEF. Here, we showed that an LysR-type transcriptional regulator, TdaA, is a positive regulator of tdaCDE gene expression and binds to the tdaC promoter region.

Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease.

Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as "plate fluorescence quencher" in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets.

Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

Molecular mechanisms underlying roseobacter-phytoplankton symbioses.

Members of the Roseobacter clade of alpha-proteobacteria are among the most abundant and ecologically relevant marine bacteria. Bacterial isolates and gene sequences derived from this taxonomic lineage have been retrieved from marine environments ranging from sea ice to open ocean mixed layer to tropical coral reefs, and in ecological niches ranging from free-living plankton to sponge symbiont to biofilm pioneer. Although roseobacters are cosmopolitan in the marine environment, their numbers and activity significantly rise with increases in the population density of phytoplankton [1,2], suggesting that these bacteria are highly adapted to engage in these symbioses. This review examines the molecules and phenotypes of roseobacters that are important in establishing and maintaining the symbioses between roseobacters and phytoplankton.

Genetic dissection of tropodithietic acid biosynthesis by marine roseobacters.

The symbiotic association between the roseobacter Silicibacter sp. strain TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda(-)) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F, could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA-DNA hybridization in 88% of a diverse collection of nine roseobacters with known antibiotic activity. These data suggest that roseobacters may use a common pathway for TDA biosynthesis that involves plasmid-encoded proteins. Using metagenomic library databases and a bioinformatics approach, differences in the biogeographical distribution between the critical TDA synthesis genes were observed. The implications of these results to roseobacter survival and the interaction between TM1040 and its dinoflagellate host are discussed.

Quantify single nucleotide polymorphism (SNP) ratio in pooled DNA based on normalized fluorescence real-time PCR.

BACKGROUND: Conventional real-time PCR to quantify the allele ratio in pooled DNA mainly depends on PCR amplification efficiency determination and Ct value, which is defined as the PCR cycle number at which the fluorescence emission exceeds the fixed threshold. Because of the nature of exponential calculation, slight errors are multiplied and the variations of the results seem too large. We have developed a new PCR data point analysis strategy for allele ratio quantification based on normalized fluorescence ratio. RESULTS: In our method, initial reaction background fluorescence was determined based upon fitting of raw fluorescence data to four-parametric sigmoid function. After that, each fluorescence data point was first subtracted by respective background fluorescence and then each subtracted fluorescence data point was divided by the specific background fluorescence to get normalized fluorescence. By relating the normalized fluorescence ratio to the premixed known allele ratio of two alleles in standard samples, standard linear regression equation was generated, from which unknown specimens allele ratios were extrapolated using the measured normalized fluorescence ratio. In this article, we have compared the results of the proposed method with those of baseline subtracted fluorescence ratio method and conventional Ct method. CONCLUSION: Results demonstrated that the proposed method could improve the reliability, precision, and repeatability for quantifying allele ratios. At the same time, it has the potential of fully automatic allelic ratio quantification.

Dual-probe assay for detection of lamivudine-resistance hepatitis B virus by real-time PCR.

A rapid and accurate minor groove binder (MGB) real-time PCR is described for detection lamivudine resistance mutations in hepatitis B virus. The real-time PCR was compared with direct Sanger sequencing in 53 clinical patients samples, the results of the real-time PCR correlate with the nucleotide sequence and the assay has the advantage of detecting a mixture of quasi-species with higher sensitivity than sequencing. As a simple, easy, rapid, accurate and high throughout method, MGB real-time PCR assay should be useful for detecting lamivudine resistance variants during lamivudine therapy in clinical settings.

[Gene cloning of rpoD and its impact on biosynthesis of antibiotics in Fluorescent pseudomonas M18].

Fluorescent pseudomonas M18, one of plant growth promoting rhizobacteria which can inhibit growth of several phytopathogenes, produces several secondary metabolites including antibiotics phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt). The rpoD gene encoding the housekeeping sigma factor sigma70 was cloned and sequenced from M18. Through sequencing and homogeneous comparison, the deduced RpoD amino acid sequence between M18 strain and Pseudomonasfluorescens CHAO shows 100% identity. It indicates that rpoD gene is very conserved in different members of fluorescent pseudomonads. The rpoD gene was placed downstream of the constitutive Ptac promoter in the shuttle vector pME6032 between E. coli and Pseudomonas fluorescens and the recombinant plasmid was introduced into M18. It was found that the time of both PCA and Plt began to accumulate was 4 and 8 hours earlier and the yield of these antibiotics was increased one and six times more respectively in M18 in comparison with the control.