Quantitative DNA Analysis Research Articles

In Vitro Regeneration of Decellularized Pig Esophagus Using Human Amniotic Stem Cells.

Decellularization of esophagus was studied using three different protocols. The sodium deoxycholate/DNase-I (SDC/DNase-I) method was the most successful as evidenced by histology and DNA quantification of the acellular scaffolds. Acellular scaffolds were further analyzed and compared with native tissue by histology, quantitative analysis of DNA, and extracellular matrix (ECM) proteins. Histologically, the SDC/DNase-I protocol effectively produced scaffold with preserved structural architecture similar to native tissue architecture devoid of any cell nucleus. ECM proteins, such as collagen, elastin, and glycosaminoglycans were present even after detergent-enzymatic decellularization. Immunohistochemical analysis of acellular scaffold showed weak expression of Gal 1, 3 Gal epitope compared with native tissue. For performing recellularization, human amnion-derived mesenchymal stem cells (MSCs) and epithelial cells were seeded onto acellular esophagus in a perfusion–rotation bioreactor. In recellularized esophagus, immunohistochemistry showed infiltration of MSCs from adventitia into the muscularis externa and differentiation of MSCs into the smooth muscle actin and few endothelial cells (CD31). Our study demonstrates successful preparation and characterization of a decellularized esophagus with reduced load of Gal 1, 3 Gal epitope with preserved architecture and ECM proteins similar to native tissue. Upon subsequent recellularization, xenogeneic acellular esophagus also supported stem cell growth and partial differentiation of stem cells. Hence, the current study offers the hope for preparing a tissue-engineered esophagus in vitro which can be transplanted further into pigs for further in vivo evaluation.

Quantitative analysis of hepatitis B virus DNA based on raman spectroscopy combined with multivariate statistical methods

Quantitation of hepatitis B virus deoxyribonucleic acid (HBV DNA) is the most accurate method for determining the infection level of hepatitis B. It is a very meaningful means of detecting the different replication states of HBV infection. In this study, HBV DNA was detected and quantified in 499 patients with hepatitis B by confocal Raman spectroscopy combined with multivariate analysis. The effective information related to HBV DNA in spectral data was extracted by different pretreatments according to the vibrational modes of different functional groups. Quantitative analysis of HBV DNA was carried out by using the spectrum in the characteristic range of 807.027–1700.29 cm−1 for spectral peak assignment analysis and using partial least squares to construct a quantitative model. The results show that the regression model is established by combining normalization pretreatment and multivariate analysis methods. The correlation coefficient (Rc) and root mean square error of calibration from the calibration set of the selected feature range are 0.9751 and 0.3881, and the correlation coefficient (Rp) of the verification set and the root mean square error of prediction from the prediction set are 0.9673 and 0.4314. respectively. In clinical medical applications, Raman spectroscopy combined with diversified analysis methods has great research potential for the quantitative detection of HBV DNA in HBV serum.

The effect of bleaching agents on the DNA analysis of bloodstains on different floor coverings.

Blood at crime scenes is one of the most significant traces of evidence in investigation proceedings. Cleaning up these traces with household cleaning products, often containing bleaching agents, inhibits or complicates the detection of DNA. In this study, human blood was applied onto different floor coverings (carpet, laminate, parquet, PVC, tile) and subsequently cleaned with water and bleaching agents (hydrogen peroxide, sodium hypochlorite, DanKlorix®, Vanish Oxi Action®) at different times. Samples have been collected afterwards from the floors. The samples underwent a quantitative and qualitative DNA analysis. Cleaning smooth surfaces with water is usually sufficed to prohibit retrieving a DNA profile in most of the cases. Cleaning carpets was more difficult due to their absorbent surface whereas the use of bleaching agents caused an additional reduction of verifiable DNA concentrations. Retrieving partial or complete profiles after the use of bleaching agents was only possible when cleaning with low concentrations of 3% hydrogen peroxide.

The Correlation between EGFR Mutation Status and DNA Content of Lung Adenocarcinoma Cells in Pleural Effusion.

Objectives: Lung adenocarcinomas with or without epidermal growth factor receptor (EGFR) mutations have shown different drug effects against EGFR inhibitors. But it is not very clear if EGFR mutation status affects the biological behavior of lung adenocarcinoma, because tumor gene regulation is very complicated and can be affected by many factors. We aimed to explore if EGFR mutation status is related with tumor malignant degree by investigating the relevance of EGFR mutation status with DNA content and aneuploid peaks of lung adenocarcinoma cells in pleural fluids without using EGFR-TKIs.Materials and Methods: 591 cases of lung adenocarcinoma patients in Hebei Tumor Hospital who had undergone EGFR gene detection and DNA quantitative analysis were collected from January 2012 to August 2018.They were divided into two groups: EGFR mutant group and non-mutant group. EGFR mutations were detected by Amplification Refractory Mutation System (ARMS) and ABI 7500 Fluorescence quantitative PCR with pleural effusions. DNA content and aneuploid peaks were detected by LD DNA image cytometry (DNA-ICM). Rank-sum test of SPSS 16 was used for statistical analysis.Results: The maximum DI, the mean DI of the first 20 cells greater than 5C, the percentage of cells greater than 5C and the number of cells greater than 9C of the first 20 cells in the mutant group were all higher than those in the non-mutant group, having statistical significance (p<0.001); the peaks of aneuploid cells in the mutant group occurred more often than those in the non-mutant group, having statistical significance (p<0.001).Conclusions: Our study has shown that advanced lung adenocarcinomas with EGFR-mutations had higher DI values, more aneuploid cells and more frequent aneuploid peaks compared with those without EGFR-mutations, suggesting that advanced lung adenocarcinomas with EGFR mutations are more aggressive than those without EGFR mutations.

The Value of DNA Quantitative Cytology Test for the Screening of Endometrial Cancer.

ObjectiveTo evaluate the accuracy, sensitivity, and specificity of DNA quantitative cytology test for the diagnosis of endometrial cancer or precancerous lesions and then discuss the value of DNA quantitative cytology as a screening tool for endometrial cancer.MethodsThe study enrolled 575 patients from September 2013 to January 2017 in Shanghai Minhang Central Hospital. Endometrial hysteroscopy plus dilation and curettage and DNA quantitative cytology tests were conducted as a method for the diagnosis of endometrial cancer. The accuracy, sensitivity, and specificity of this method were calculated according to histopathologic diagnoses which were used as the gold standard for diagnosis confirmation.ResultsFor the DNA quantitative cytology diagnosis of endometrial cancer, accuracy was estimated at 85.57%, sensitivity at 87.01%, specificity at 85.34%, positive predictive value (PPV) at 47.86%, and negative predictive value (NPV) at 97.07%. For the DNA quantitative cytology diagnosis of endometrial cancer in menopausal patients: accuracy was estimated at 89.95%, sensitivity at 97.73%, specificity at 87.59%, positive predictive value (PPV) at 70.49%, negative predictive value (NPV) at 99.22%. For the DNA quantitative cytology diagnosis of endometrial cancer in non-menopausal patients, accuracy was estimated at 83.42%, sensitivity at 72.73%, specificity at 84.42%, positive predictive value (PPV) at 30.38%, and negative predictive value (NPV) at 97.07%.ConclusionDNA heteroploidy can be tested for the occurrence and the development of endometrial cancer. A small number of non-endometrial cancer cases may also appear DNA heteroploidy, but the number of >5c cells is less than 3. DNA quantitative analysis is a useful tool for the screening of endometrial cancer, worthy of being popularized and applied in endometrial cancer diagnosis.

Assessing human-specific CrAssphage recovery after acidification-filtration concentrating method in environmental water.

Pinpointing water pollution sources using host-specific gastrointestinal microbes, known as microbial source tracking (MST), have significant benefits for countries with water quality management issues related to pollution. A recently discovered bacteriophage, crAssphage, shows promise as a human-specific MST marker. However, loss of genetic materials during the recovery and the detection processes could alter the ability to measure virus quantities in a water sample. This study determined the crAssphage recovery efficiencies in water sources, including seawater, freshwater, and influent and effluent from a wastewater treatment plant, by spiking natural crAssphage concentrates prior to DNA extraction and quantitative PCR analysis. The results showed that river and seawater with no or low crAssphage background experienced no recovery loss. Evaluating recovery efficiencies in samples with high crAssphage backgrounds posed a challenge due to the inability to prepare high crAssphage titers. This study highlights the importance of intra-laboratory assessment of recovery efficiency in environmental samples for retrieving absolute crAssphage quantification with correction of bias among water samples and increase in data accuracy. PRACTITIONER POINTS: In laboratory assessment of recovery efficiency is crucial for bias correction and data accuracy for absolute crAssphage quantification in water samples. No loss in crAssphage recovery was observed in river and seawater that contained no or low crAssphage backgrounds. Inability to prepare high crAssphage spike concentrations remains the major limitation for evaluating recovery in samples with high crAssphage backgrounds. The results underline the importance of evaluating method recovery in real environmental samples that reflect actual matrix effect. Absolute crAssphage quantification, as human-specific pollution marker, could be used for prioritizing water quality restoration and area-based management plan.

Overexpression of MfSAMS1 Improves Chilling Tolerance in Transgenic Stylo

Stylo [Stylosanthes guianensis (Aublet) Sw.] is an important forage legume and cover crop in tropical and southern subtropical regions. Its growth and survival are largely affected by low temperature in winter in subtropical regions. It is important to improve its chilling tolerance. S‐adenosylmethionine synthetase (SAMS) is the key enzyme catalyzing the formation of S‐adenosylmethionine (SAM), a precursor of polyamines synthesis. To improve chilling tolerance in stylo, MfSAMS1 from Medicago falcata was overexpressed in stylo plants. DNA gel blot hybridization and quantitative reverse transcription PCR analysis showed that MfSAMS1 was inserted into the genomes of transgenic plants and expressed. Compared to the wild‐type, transgenic plants had enhanced SAM and spermidine levels and increased chilling tolerance with higher level of the maximum photochemical efficiency of photosystem II (Fv/Fm) and lower ion leakage after chilling treatment. Polyamine oxidase activity and H2O2 levels were increased in transgenic plants compared with the wild‐type. In addition, higher activities of superoxide dismutase and catalase were observed in transgenic plants than in the wild‐type. The results suggest that overexpression of MfSAMS1 promoted polyamine synthesis and oxidation, which in turn improved H2O2–induced antioxidant protection and protected transgenic plants against chilling stress.Core Ideas Transgenic stylo plants overexpressing MfSAMS1 were produced. Plants showed promoted polyamine synthesis and oxidation. Transgenic plants had increased chilling tolerance with higher antioxidant levels.

Диагностика и лечение абактериаль-ного ХП/СХТБ III, ассоциированного с герпесвирусами

The etiology of abacterial CP/CPPS (category III) has not been studied enough. Currently, there is no gold standard of diagnostic study and optimal treatment algorithm. The aim of our study was to study three human herpes viruses (HHV) in clinical samples from patients with inflammatory diseases of urogenital tract and to evaluate the efficiency of proposed treatment algorithm for abacterial CP/CPPS. The biological samples from the urogenital tract (urethral swab, ejaculate and expressed prostatic secretions) from 101 patients with category III CP/CPPS were studied. Quantitative analysis of HHV DNA (CMV, EBV and HHV-6) was performed by PCR. HHV DNA was detected in 38/101 patients (37.6%) in Group 1. Among the detected viral types, HHV-6 was the most common (52%). Analysis of biological samples form the three sources revealed that viral DNA was determined in urethral swab in concentration of 3,703,900 copies/ml. In Group 2, viral DNA was not detected in 63 patients. Evaluation of results of the standard treatment in HHV-negative patients (n=63) and antibiotic-free scheme, including the immunoregulatory drug Viferon, in HHV-positive patients (n=38) showed that the number of HHV-positive samples after treatment decreased by 54.3%. In addition, severity of all symptoms according to NIH-CPSI scale significantly decreased in both groups (p<0.0001). There was an improvement in all clinical symptoms in Group 1 by 47.9%, especially for pain + urination (52%). It should be noted that a positive response to treatment, which was confirmed by the changes in total score of NIH-CPSI scale, was noted in all patients in Group 1. Detection of herpes viruses in the urogenital tract of patients with abacterial CP/CPPS suggests possible role of viral infections in its etiology. The comparative analysis of the results of standard treatment including antiviral, immunomodulatory and antioxidant drugs showed that the use of complex therapy without antibiotics allowed to eliminate or significantly reduce the concentration of viruses in urogenital tract, as well as significantly reduce the clinical manifestations of abacterial CP/CPPS.

A novel data processing method CyC* for quantitative real time polymerase chain reaction minimizes cumulative error.

Quantitative real-time polymerase chain reaction (qPCR) is routinely conducted for DNA quantitative analysis using the cycle-threshold (Ct) method, which assumes uniform/optimum template amplification. In practice, amplification efficiencies vary from cycle to cycle in a PCR reaction, and often decline as the amplification proceeds, which results in substantial errors in measurement. This study reveals the cumulative error for quantification of initial template amounts, due to the difference between the assumed perfect amplification efficiency and actual one in each amplification cycle. The novel CyC* method involves determination of both the earliest amplification cycle detectable above background (“outlier” C*) and the amplification efficiency over the cycle range from C* to the next two amplification cycles; subsequent analysis allows the calculation of initial template amount with minimal cumulative error. Simulation tests indicated that the CyC* method resulted in significantly less variation in the predicted initial DNA level represented as fluorescence intensity F0 when the outlier cycle C* was advanced to an earlier cycle. Performance comparison revealed that CyC* was better than the majority of 13 established qPCR data analysis methods in terms of bias, linearity, reproducibility, and resolution. Actual PCR test also suggested that relative expression levels of nine genes in tea leaves obtained using CyC* were much closer to the real value than those obtained with the conventional 2-ΔΔCt method. Our data indicated that increasing the input of initial template was effective in advancing emergence of the earliest amplification cycle among the tested variants. A computer program (CyC* method) was compiled to perform the data processing. This novel method can minimize cumulative error over the amplification process, and thus, can improve qPCR analysis.

Reconstructed Skin Models Revealed Unexpected Differences in Epidermal African and Caucasian Skin

Clinical observations of both normal and pathological skin have shown that there is a heterogeneity based on the skin origin type. Beside external factors, intrinsic differences in skin cells could be a central element to determine skin types. This study aimed to understand the in vitro behaviour of epidermal cells of African and Caucasian skin types in the context of 3D reconstructed skin. Full-thickness skin models were constructed with site matched human keratinocytes and papillary fibroblasts to investigate potential skin type related differences. We report that reconstructed skin epidermis exhibited remarkable differences regarding stratification and differentiation according to skin types, as demonstrated by histological appearance, gene expression analysed by DNA microarray and quantitative proteomic analysis. Signalling pathways and processes related to terminal differentiation and lipid/ceramide metabolism were up-regulated in epidermis constructed with keratinocytes from Caucasian skin type when compared to that of keratinocytes from African skin type. Specifically, the expression of proteins involved in the processing of filaggrins was found different between skin models. Overall, we show unexpected differences in epidermal morphogenesis and differentiation between keratinocytes of Caucasian and African skin types in in vitro reconstructed skin containing papillary fibroblasts that could explain the differences in ethnic related skin behaviour.

A Pipeline for Faecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR

Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a bio-specimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics (i.e., Bristol scores). We further extended this approach to mouse stool analysis, to enable faecal host DNA studies in animal disease models as well.

Bases are Not Letters: On the Analogy between the Genetic Code and Natural Language by Sequence Analysis

The article deals with the notion of the genetic code and its metaphorical understanding as a “language”. In the traditional view of the language metaphor of the genetic code, combinations of nucleotides are signs of amino acids (see the table of the genetic code). Similarly, words combined from letters (speech sounds) represent certain meanings. The language metaphor of the genetic code (Markos and Faltýnek, Biosemiotics 4(2), 171–200, 2011) assumes that the nucleotides stay in the analogy to letters, triples to words and genes to sentences (Jakobson 1971). We propose an application of mathematical linguistic methods on the notion of the genetic code. We provide quantitative analysis (n-gram structure, Zipf’s law) of mRNA strings and natural language texts. This analysis is sensitive to the detection of the code (language) units hierarchy. We also take into consideration a representative quantitative analysis of DNA, RNA and proteins. Our analysis of mRNA confirms an assumption that the design of the genetic code cannot analogize DNA bases and letters. The notion of the letter is much more appropriate if analogized with triplets or amino acids (see Lackova et al, Theory in Biosciences 136(3–4), 187–191, 2017).

P212 - Quantitative analysis of plasma cell-free DNA and its DNA integrity in patients with metastatic breast cancer using ALU sequence

P212 - Quantitative analysis of plasma cell-free DNA and its DNA integrity in patients with metastatic breast cancer using ALU sequence

A highly integrated real-time digital PCR device for accurate DNA quantitative analysis

Misclassification of positive partitions in microfluidic digital polymerase chain reaction (dPCR) can cause the false positives and false negatives, which significantly alter the resulting estimate of target DNA molecules. To address this issue, establishing real-time fluorescence interrogation of each partition in microfluidic arrays is an effective way in which false positive and false negative partitions can be eliminated. However, currently available devices for real-time fluorescence interrogation are either not competent for microfluidic digital array, or they are bulky, expensive and entail peripheral equipment due to low integration. Therefore, in this study, a Raspberry Pi based, low-cost and highly integrated device is presented to achieve real-time fluorescence detection for microfluidic digital array, termed real-time dPCR device. In the device, uniform thermocycler, streamlined real-time fluorescence imaging setup, and compact data processing system are all integrated to undergo on-chip dPCR amplification, real-time fluorescence detection, and data analysis. Using this real-time dPCR device, the accuracy of DNA absolute quantification by dPCR is improved, since the misclassification of positive partitions is efficiently reduced based on the characteristic real-time fluorescence curves of positive partitions in a self-priming microfluidic chip. Compared with end-point dPCR on our device and commercialized QuantStudio™ 3D dPCR system, the real-time dPCR on our device exhibits a higher accuracy for DNA quantification. In addition, this real-time dPCR device is much smaller and cheaper than the commercialized Digital PCR system, but not sacrificing the capability of error correction for absolute quantitation analysis. Conclusively, this highly integrated real-time dPCR device is very beneficial for DNA quantitative analysis where the determination accuracy is pivotal.

An enzymatic paper-based biosensor for ultrasensitive detection of DNA.

Point-of-care Nucleic acid testing (POCNAT) has become an attractive technique for DNA identification in resource-limited settings, offering a rapid system for urgent clinical applications. In this study, a chemiluminescence-based lateral flow biosensor (CL-LFB) was developed for the quantitative analysis of DNA, without labeling and amplification. The developed biosensor employs a two-step hybridization, a primary hybridization of 5'-biotinylated detector probe to the target DNA and a secondary hybridization of the resulting complex with the immobilized capture probe. Quantitative analysis of DNA was provided via HRP-catalyzed reaction with the chemiluminescense substrate followed by imaging with a complementary metal-oxide-semiconductor (CMOS) digital camera. The assay performance was investigated using a synthetic target, 16S rRNA gene (775 bp) and the whole genome derived from Escherichia coli (E.coli). A detection limit of 1.5 pM for the synthetic target and 0.4 ng/ml for 16S rRNA gene was obtained. In spite of LFBs limitations for the detection of large DNA fragments, the proposed assay provided a low-cost, fast, and sensitive tool for PCR-free diagnosis of small and larger fragments of nucleic acids.

Biochemical and Molecular Studies Related to Phase Change in Gregarious and Solitarious Desert Locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae)

Quantitative and qualitative analysis of protein and DNA were investigated in two extreme phases, gregarious and solitarious Schistocerca gregaria, in addition to, their transient stages. The level of protein in long-term solitarious S. gregaria revealed highly significant reduction when compared with the gregarious phase (P<0.05). These protein levels were 16.33±0.14 and 7.95±0.21 mg/10ml for long-term gregarious and solitarious S. gregaria, respectively. Data revealed 52 bands as the maximum number of protein which detected at the molecular weight (Mw) ranged from 25 to 225.4 kilodaltons (kDa). The most separated protein bands were detected in Mw ranging from 46 to 25 kDa. Likewise, quantitative analysis of DNA levels showed a significant reduction of DNA level in long-term solitarious S. gregaria, compared with gregarious phase (P<0.05). These DNA levels were 7.75± 0.30 and 4.4±0.28 µg/µl for long-term gregarious and solitarious S. gregaria, respectively. Furthermore, the fraction DNA pattern of tissue assessed that there are 15 different DNA bands detected at Mw ranging from 366 to 3484 base pair (bp). In conclusion, each phase of S. gregaria has a significant change in protein content and the fraction pattern of the DNA, which was clearly reflected in genetic and epigenetic differences among different phases of S. gregaria.

Atrazine hinders PMA-induced neutrophil extracellular traps in carp via the promotion of apoptosis and inhibition of ROS burst, autophagy and glycolysis

Atrazine (ATR), a selective herbicide, is consistently used worldwide and has been confirmed to be harmful to the health of aquatic organisms. The release of neutrophil extracellular traps (NETs) is one of the newly discovered antimicrobial mechanisms. Although several immune functions have been analyzed under ATR exposure, the effect of ATR on NETs remains mainly unexplored. In the present study, we treated carp neutrophils using 5 μg/ml ATR and 5 μg/ml ATR combined with 100 nM rapamycin to elucidate the underlying mechanisms and to clarify the effect of ATR on phorbol myristate acetate (PMA)-induced NETs. The results of the morphological observation and quantitative analysis of extracellular DNA and myeloperoxidase (MPO) showed that NETs formation were significantly inhibited by ATR exposure. Moreover, we found that in the NETs process, ATR downregulated the expression of the anti-apoptosis gene B-cell lymphoma-2 (Bcl-2), increased the expression of the pro-apoptosis factors Bcl-2-Associated X (BAX), cysteinyl aspartate specific proteinases (Caspase3, 9), and anti-autophagy factor mammalian target of rapamycin (mTOR), decreased the expression of autophagy-related protein light chain 3B (LC3B) and glucose transport proteins (GLUT1, 4), disturbed the activities of phosphofructokinase (PFK), pyruvate kinase (PKM), and hexokinase (HK) and limited reactive oxygen species (ROS) levels, indicating that the reduced NETs release was a consequence of increased apoptosis and diminished ROS burst, autophagy and down-regulated glycolysis under ATR treatment. Meanwhile, rapamycin restored the inhibited autophagy and glycolysis and thus resisted the ATR-suppressed NETs. The present study perfects the mechanism theory of ATR immunotoxicity to fish and has a certain value for human health risk assessment.

Smartphone-based mobile digital PCR device for DNA quantitative analysis with high accuracy

Digital polymerase chain reaction (dPCR) circumventing the external calibration and potentially providing absolute quantification of nucleic acids has become an increasingly popular manifestation of PCR in biological researches. However, currently reported or commercial dPCR devices are not suitable for applications in laboratories or zones with limited infrastructures, due to low function integration, cost-inefficiency, or weak mobility. Herein, in order to enable accurate DNA quantitative analysis in such situations, we have developed a smartphone-based mobile dPCR device integrated with thermal cycling control, on-chip dPCR, data acquisition, and result analysis. All the function units are automatically controlled using a customized Android software. The device is approximately 90 mm × 90 mm × 100 mm in size and about 500 g in weight, only costing about 320 dollars except the smartphone. Coupled with the self-priming dPCR chip previously developed by our lab, the device is able to accurately quantify down to 10 copies of the human 18 S ribosomal DNA fragment inserted in a plasmid. Comparing to the commercial QuantStudio™ 3D dPCR platform, our device achieves a comparable analytical accuracy. Besides, our device is capable of detecting single molecule of cancer biomarker gene CD147 in a low number of HepG2 cells. Therefore, our dPCR device as a low-cost, potable, and robust tool for highly accurate DNA quantitative analysis has a great potential in Point-of-care (POC) applications.

Establishment of COS-JC cells persistently producing archetype JC polyomavirus.

JC polyomavirus (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunocompromised patients. Archetype JCPyV circulates in the human population. There have been several reports of archetype JCPyV replication in cultured cells, in which propagation was not enough to produce high titers of archetype JCPyV. In this study, we carried out cultivation of the transfected cells with archetype JCPyV DNA MY for more than 2 months to establish COS-7 cells (designated COS-JC cells) persistently producing archetype JCPyV. Moreover, JCPyV derived from COS-JC cells was characterized by analyzing the viral propagation, size of the viral genome, amount of viral DNA, production of viral protein, and structure of the non-coding control region (NCCR). Southern blotting using a digoxigenin-labeled JCPyV probe showed two different sizes of the JCPyV genome in COS-JC cells. For molecular cloning, four of five clones showed a decrease in the size of complete JCPyV genome. Especially, clone No. 10 was generated the large deletion within the Large T antigen. On the other hand, the archetype structure of the NCCR was maintained in COS-JC cells, although a few point mutations occurred. Quantitative PCR analysis of viral DNA in COS-JC cells indicated that a high copy number of archetype JCPyV DNA was replicated in COS-JC cells. These findings suggest that COS-JC cells could efficiently propagate archetype JCPyV MY and offer a useful tool to study persistent infection of archetype JCPyV in a kidney-derived system.

Effects of Sucrose vs. Fructose-and-Glucose Intakes on Gut Abundance of Akkermansia Muciniphila and Serum Kidney Injury Molecule-1 in Rats

Increase of obesity and chronic kidney disease (CKD) in U.S. coincided with replacing sucrose (S) with HFCS: blend of 55% fructose (F) and 42% glucose (G). Both obesity and CKD associate with gut dysbiosis. Given that S is not utilized by all gut bacteria, microbiota may differ with ingestion of free or bound F and G. We aimed to compare effects of S vs. F and G on the gut contents of Akkermansia muciniphila-bacterium reported to improve host metabolic health, and serum Kidney Injury Molecule-1 (KIM-1), tubular injury marker shown to predict CKD. Male 4-week old Sprague-Dawley rats were assigned to 16 weeks of unlimited access to standard chow and 5% solution of S (S group) or 5% solution of 55%F and 45%G blend (55F/45G) or tap water (W). Fluid intake and urine volume were higher in S or 55F/45G rats and F accounted for ∼10% of their caloric intake. Interestingly, in these animals serum triglyceride concentration was lower, while serum total protein and albumin were higher than in controls (table). On the other hand, serum KIM-1 was significantly lower and fecal A. muciniphila much more abundant in S than in 55F/45G or W. Intake of S solution in rats, as opposed to free F and G or just regular chow, increases the gut contents of A. muciniphila and appears to improve condition of renal tubulointerstitium. If present in humans, this may account for obesity and CKD increase following S replacement by HFCS. S (n=20)55F/45G (n=17)W (n=16)Final body weight [g] 24h urine volume [mL]444.1±30.6 40.4±46.2^425.2±41.7 22.3±24.6^457.9±66.3 9.5±3.5Serum glucose [mmol/L] Serum uric acid [μmol/L]7.58±1.05 43.1±10.27.47±1.12 45.0±16.77.82±0.75 49.4±20.5Serum cholesterol [mmol/L] Serum triglycerides [mmol/L]1.02±0.13 0.31±0.14^1.06±0.22 0.29±0.18^1.02±0.13 0.46±0.17Serum total protein [g/dL] Serum albumin [g/dL]6.95±0.41^ 1.96±0.14^6.74±0.41^ 1.93±0.11^6.30±0.37 1.71±0.11Serum KIM-1 [pg/mL]1162.8±34.0*^212.0±64.3218.3±43.7Fecal Akkermansia muciniphila [%]22.30±2.57*^0.14±0.230.64±1.49Kruskal-Wallis ANOVA with Mann-Whitney U test as post-hoc: *P&amp;lt;0.vs. 55F/45G; ^P&amp;lt;0.vs. W 1serum KIM-1 assessed with ELISA (R&amp;D Systems) 2gut flora composition assessed with quantitative analysis of fecal DNA encoding bacterial 16S rRNA (Illumina HiSeq) Disclosure G. Wystrychowski: Employee; Self; DaVita Poland. E. Pikulska-Chrobak: None. J. Wojcik: None. M. Kozubska: None. W. Trautsolt: Other Relationship; Self; Bayer AG, Novo Nordisk A/S. S. Gorczynska-Kosiorz: Other Relationship; Self; Bayer AG, Novo Nordisk A/S. R. Kuźniewicz: Other Relationship; Self; Roche Pharma. E. Zukowska-Szczechowska: Employee; Self; Fresenius Medical Care. W. Grzeszczak: Speaker's Bureau; Self; Boehringer Ingelheim GmbH, Bioton, Merck &amp; Co., Inc., Novo Nordisk A/S. Stock/Shareholder; Self; Eli Lilly and Company. Speaker's Bureau; Self; Bayer AG. A. Wystrychowski: Other Relationship; Self; Roche Pharma.