Specific Fragments Research Articles

Ensemble hologram quantitative structure activity relationship model of the chromatographic retention index of aldehydes and ketones

色谱保留指数(retention index, RI)是色谱分析中的重要参数,不同化合物在不同极性固定相上具有不同的保留行为。醛酮化合物种类众多,实验测定其RI值的时间和经济成本高。该论文采用集成建模(ensemble modeling)结合全息定量构效关系(HQSAR)方法研究了醛酮化合物在2种固定相(DB-210和HP-Innowax)上色谱保留指数的定量构效关系(QSAR)模型。用外部测试集验证法和留一交叉验证法评估了所建立模型的预测能力。首先建立了34种被研究化合物的个体HQSAR模型。在固定相DB-210上,片段特性(FD)为“供体/受体原子(DA)”且片段尺寸(FS)为1~9时可得到最优个体模型,在固定相HP-Innowax上,FD为“DA”且FS为4~7时可得到最优个体模型,这两个模型的交叉验证相关系数( )分别为0.935和0.909,外部验证相关系数( )分别为0.925和0.927,一致性相关系数(CCC)分别为0.953和0.960,预测平方相关系数F2( )分别为0.922和0.918,预测平方相关系数F3( )分别为0.931和0.927。研究结果表明醛酮化合物的分子结构与RI值之间存在定量关系,用HQSAR方法可以建立二者之间的QSAR模型。其次,以4个预测准确度最高的个体HQSAR模型作为子模型通过算术平均建立了集成HQSAR模型。建立的集成HQSAR模型预测被研究化合物在DB-210和HP-Innowax固定相上RI值的 分别为0.927和0.919, 分别为0.929和0.963, CCC分别为0.956和0.979, 分别为0.927和0.958, 分别为0.935和0.963。与个体HQSAR模型相比,建立的集成HQSAR模型预测准确度更高。这说明集成建模是提高HQSAR模型预测能力的有效方法,HQSAR与集成建模方法相结合可以用于研究和预测醛酮化合物的RI值。

Evaluation of Time-of-Flight Secondary Ion Mass Spectrometry Spectra of Peptides by Random Forest with Amino Acid Labels: Results from a Versailles Project on Advanced Materials and Standards Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the identification of peptide sample TOF-SIMS spectra by machine learning. More than 1000 time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra of six peptide model samples (one of them was a test sample) were collected using 27 TOF-SIMS instruments from 25 institutes of six countries, the U. S., the U. K., Germany, China, South Korea, and Japan. Because peptides have systematic and simple chemical structures, they were selected as model samples. The intensity of peaks in every TOF-SIMS spectrum was extracted using the same peak list and normalized to the total ion count. The spectra of the test peptide sample were predicted by Random Forest with 20 amino acid labels. The accuracy of the prediction for the test spectra was 0.88. Although the prediction of an unknown peptide was not perfect, it was shown that all of the amino acids in an unknown peptide can be determined by Random Forest prediction and the TOF-SIMS spectra. Moreover, the prediction of peptides, which are included in the training spectra, was almost perfect. Random Forest also suggests specific fragment ions from an amino acid residue Q, whose fragment ions detected by TOF-SIMS have not been reported, in the important features. This study indicated that the analysis using Random Forest, which enables translation of the mathematical relationships to chemical relationships, and the multi labels representing monomer chemical structures, is useful to predict the TOF-SIMS spectra of an unknown peptide.

Preparation of transgenic Musca domestica by microinjection method

The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.

A Low-Cost SARS-CoV-2 rRBD ELISA to Detect Serostatus in Ecuadorian Population with COVID-19.

ABSTRACTLaboratory diagnosis of the COVID-19 relies on RT-PCR to amplify specific fragments of SARS-CoV-2 genome. However, serological tests are required to determine the immune response elicited after infection. Here, we analyzed convalescent sera collected from positive individuals by RT-PCR to SARS-CoV-2 (n = 78), Zika (n = 20), dengue (n = 20), chikungunya (n = 54), intestinal parasites (n = 11), and HIV (n = 1), from different areas of Ecuador, with an in-house ELISA using a SARS-CoV-2 receptor binding domain recombinant (rRBD) antigen to detect IgG antibodies elicited by SARS-CoV-2 infection. Of the 78 samples positive for SARS-CoV-2 by RT-PCR, 73 showed high absorbance value compared with the cutoff and five were negative. All tested sera from other infections showed no reactivity. Sensitivity, specificity, positive predictive value, and negative predictive value were 93.6%, 100%, 100%, and 95.4%, respectively. This in-house anti-IgG rRBD ELISA offers an economic and simple alternative to determine IgG immune responses after SARS-CoV-2 infection.

Functional analysis of CgWRKY57 from Cymbidium goeringii in ABA response.

BackgroundThe orchid is one of the top ten Chinese flowers and has high ornamental value and elegant color. However, orchids are vulnerable to abiotic stresses during their growth and development, and the molecular mechanism of the abiotic stress response in orchids is unclear. WRKY proteins belong to a transcription factor family that plays important roles in biotic stress, abiotic stress, growth and development in plants, but little is known about the WRKY family in Cymbidium goeringii.MethodsThe specific fragment of the CgWRKY57 gene of C. goeringii was analyzed by bioinformatics. The expression of the CgWRKY57 gene of C. goeringii under 4 °C, 42 °C water and ABA stress as well as different tissues was detected by real-time fluorescence quantitative PCR. CgWRKY57 gene was overexpressed in wild type Arabidopsis thaliana by inflorescence infection method, and the function of transgenic lines under ABA stress was analyzed.ResultsCgWRKY57 was cloned from C. goeringii and found to encode 303 amino acids. The CgWRKY57 protein is an acidic, nonsecreted hydrophilic protein without a signal peptide or transmembrane domain. The CgWRKY57 protein is located to the nucleus and may function intracellularly according to its predicted subcellular localization. A domain analysis and homology comparison showed that the CgWRKY57 protein has a “WRKYGQK” domain and belongs to Group III of the WRKY family, and a phylogenetic analysis demonstrated that CgWRKY57 is closely related to OsWRKY47. CgWRKY57 was expressed in the roots, stems, leaves and floral organs of C. goeringii, and its expression level was highest in the roots according to real-time qPCR analysis. There were significant differences in CgWRKY57 expression under 4 °C, 42 °C ABA and water stress treatments, and its expression changed greatly under ABA stress. The expression of CgWRKY57 in transgenic plants was significantly higher than that in wild type plants under ABA stress, and the root length and germination rate were reduced in transgenic plants compared to wild type plants.ConclusionsThese results indicate that CgWRKY57 overexpression is responsive to ABA stress, and they provide a foundation for future analyses of the biological functions of the WRKY family in C. goeringii.

Novel analysis procedure for red ginseng polysaccharides by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry

BackgroundRed ginseng polysaccharides (RGPs) have been acknowledged for their outstanding immunomodulation and anti-tumor activities. However, their studies are still limited by the complexity of their structural features, the absence of purification and enrichment methods, and the rarity of the analytical instruments that apply to the analysis of such macromolecules. Thus, this study is an attempt to establish a new mass spectrometry (MS)-based analysis procedure for RGPs. MethodsSaponin pre-excluded powder of RG (RG-SPEP, 10 mg) was treated with 200 μL of distilled water and centrifuged for 5 h at 1000 rpm and 85 °C. Ethanol-based precipitation and centrifugation were applied to obtain RGPs from the heated extracts. Further, endo-carbohydrase treatments were performed to produce specific saccharide fragments. Solid-phase extraction (SPE) processes were implemented to purify and enrich the enzyme-treated RGPs, while matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) MS was employed for the partial structural analysis of the obtained RGPs. ResultsUtilizing cellulase, porous graphitized carbon (PGC), hydrophilic interaction chromatography (HILIC), and MALDI-TOF/TOF MS, the neutral and acidic RGPs were qualitatively analyzed. Hexn and Hexn−18 (cellulose analogs) were determined to be novel neutral RGPs. Additionally, the [Unknown + Hexn] species were also determined as new acidic RGPs. Furthermore, HexAn (H) was determined as another form of the acidic RGPs. ConclusionCompared to the previous methods of analysis, these unprecedented applications of HILIC-SPE and MALDI-TOF/TOF MS to analyze RGPs proved to be fairly effective for fractionating and detecting neutral and acidic components. This new procedure exhibits great potential as a specific tool for searching and determining various polysaccharides in many herbal medicines.

Abstract PS3-04: Breast tumor targeted fluorescence guidance for intraoperative margin assessment

Abstract BACKGROUND: A majority of the 270,000 women diagnosed with breast cancer in the United States annually are candidates for breast conserving surgery (lumpectomy), however, up to 30% of patients require reoperation due to positive margins. The ideal resolution is to make residual in-breast disease visible at the time of surgery. The goal of this research is to develop a systemically administered fluorescent molecular imaging agent that is targeted to breast tumors for intraoperative margin assessment using fluorescence guided surgery (FGS). METHODS: To identify targetable cell-surface markers for use in FGS, gene expression profiling of publicly available Affymetrix mRNA microarray data from patients with invasive ductal or invasive lobular carcinoma tumors (n=34) and surrounding normal breast tissues (n=32) was performed. Protein expression of select targets was determined by immunohistochemistry (IHC) staining of patient tissue specimens. A humanized CEACAM6 (carcinoembryonic cell adhesion molecule 6) specific antibody fragment (scFv-Fc [IgG4]) was conjugated to the near-infrared IRDye800CW fluorescent dye (LiCor) by NHS-ester linkage to create CEACAM6-800. The conjugate was characterized for in vitro specificity by immunocytochemistry (ICC) and live cell uptake studies. As proof-of-principle, the conjugate (15 µg) was intravenously injected into nude mice bearing orthotopic human MCF-7 breast cancer mammary fat pad (MFP) xenograft tumors with endogenous expression of CEACAM6. FGS was performed using the “SurgVision” clinical imaging platform (approved for clinical use in Europe) at 24 h post-injection.RESULTS: We identified 263 genes with higher mRNA expression in tumor relative to normal. From this list, we selected 9 genes for confirmation of protein expression by IHC. Of the 9 targets, only CEACAM6 had no protein expression in normal breast tissues and robust expression in ~50% of tumors. ICC staining using the CEACAM6-800 conjugate and multiple human ductal carcinoma tumor cell lines confirmed the cell surface localization of CEACAM6. In live-cell uptake studies, CEACAM6-800 accumulated on the cell surface as early as 10 min after addition to the media and was internalized after 90 min. Blocking studies demonstrated specificity. In the proof-of-principal FGS study, we observed high fluorescence in MCF-7 MFP tumors relative to background and the tumors and their residuals were completely removed in the first attempt. CONCLUSION: We have confirmed CEACAM6 protein as a cell-surface adhesion receptor target for breast cancer FGS. The CEACAM6-800, fluorescent-dye to antibody-fragment conjugate was prepared and tested in vitro for sub-cellular localization and specific uptake. In vivo studies demonstrated specific uptake into MFP tumors and FGS for tumor resection. We are currently performing studies to determine the in vivo pharmacokinetics of CEACAM6-800 uptake and clearance in tumor and normal tissues and pre-clinical post-surgery survival using our pre-clinical models. These studies will enable future translational studies. Citation Format: Narges Tafreshi, Mikalai Budzevich, Weihong Sun, Daruka Mahadevan, Marie Catherine Lee, David L Morse. Breast tumor targeted fluorescence guidance for intraoperative margin assessment [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-04.

Abstract PD9-02: Personalized ctDNA as a predictive biomarker in high-risk early stage breast cancer (EBC) treated with neoadjuvant chemotherapy (NAC) with or without pembrolizumab (P)

Abstract Background: In the I-SPY 2 TRIAL, the addition of P to standard NAC resulted in more than doubling of the pathologic complete response (pCR) rates for both hormone receptor-positive (HR+)/HER2- and triple-negative (TN) early breast cancer (EBC) patients (pts) compared to NAC only (Nanda et al, JAMA Oncol, 2020). At 3 years, distant recurrence-free survival (DRFS) rates in pts with pCR following NAC+P was >95%. We hypothesized that ctDNA can serve as a predictive biomarker of response and survival in pts treated with NAC. Methods: A personalized ctDNA test (Signatera) was performed on 511 serial plasma samples from 138 pts with high-risk HR+/HER2- (n=77) or TN (n=61) stage II/III EBC. Pts received P with paclitaxel (Tx) followed by AC (P arm, n=42) or standard NAC only (n=96), an exploratory subset of pts evaluated for P efficacy. Plasma was collected; pretreatment (T0), 3 weeks after treatment initiation (T1), between Tx+/-P and AC regimens (T2), and prior to surgery (T3). ctDNA was deemed positive with a minimum of 2 of the pt specific tumor mutation fragments detected in cfDNA. Association of ctDNA with response and survival was analyzed using logistic and Cox regressions with pCR and DRFS as endpoints. Median follow-up was 2.8 years. Results: Detection of ctDNA decreased over time (P arm: T0-81%, T1-50%, T2-19%, T3-3%) and NAC only: T0-82%, T1-65%, T2-26%, T3-10%). ctDNA data at T0 and T1 was available for 96% (132/138) of pts in P arm or NAC only (Table). Among ctDNA+ patients at baseline, clearance at T1 was significantly associated with pCR (OR=1.92, ctDNA+/-; OR=0.27, ctDNA+/+; LR p<0.001). This association remained significant after adjustment for HR status and treatment (LR p<0.001) and P arm or NAC only (P: LR p=0.03; NAC: LR p=0.01). ctDNA data at T0, T1, and T2 was available for 86% (118/138) pts. (Table). Among all ctDNA+ pts at baseline, dynamics through T2 was associated with pCR (OR=1.44, ctDNA+/-/-; OR=0.33, ctDNA+/+/-, OR=0.12, ctDNA+/+/+; LR p=0.0011). This association remained significant when adjusted for HR status and treatment (LR p<0.001). Analysis within individual treatments showed significant association for NAC (LR p=0.040) and a non-significant trend in NAC+P (LR p=0.063), likely due to smaller sample size. All pts who achieved pCR were ctDNA- at T3 (n=34). Among those who failed to achieve pCR (n=81), DRFS was significantly better in ctDNA- (n=72/81; 20 in P and 52 in NAC) versus ctDNA+ pts (n=9/81; 1 in P and 8 in NAC) (adjusted HR 0.13; 95% CI 0.05-0.37). Conclusions: These exploratory results align with our previous findings that early clearance of ctDNA during NAC treatment was significantly associated with increased likelihood of achieving pCR. Additionally, we show that ctDNA clearance can be an early surrogate marker for therapy response assessment. Residual ctDNA after neoadjuvant treatment was a significant predictor of metastatic recurrence and death. Personalized monitoring of ctDNA during the course of NAC is feasible and provides information that can be combined with imaging and pathology, and may help to optimize decision making for de-escalation or escalation of therapy. Larger studies are ongoing. ctDNA dynamics and pCRctDNA status at T0 and T1 (n=132)ctDNA status at T0, T1, and T2 (n=118)ctDNA-/-ctDNA+/-ctDNA+/+ctDNA-/-/-ctDNA+/-/-ctDNA+/+/-ctDNA+/+/+Total, n (%)24 (18)28 (21)80 (61)22 (19)24 (20)43 (36)27 (23)pCR, n (%)9 (38)15 (54)11 (14)9 (41)12 (50)8 (19)2 (7)No pCR, n (%)15 (63)13 (46)69 (86)13 (59)12 (50)35 (81)25 (93) Citation Format: Mark Jesus M Magbanua, Denise Wolf, Derrick Renner, Svetlana Shchegrova, Lamorna Brown Swigart, Christina Yau, Gillian Hirst, Hsin-Ta Wu, Ekaterina Kalashnikova, Antony Tin, Amy Delson, Douglas Yee, Angela DeMichele, Raheleh Salari, Angel Rodriguez, Bernhard Zimmermann, Himanshu Sethi, Alexey Aleshin, Paul Billings, Laura Esserman, Minetta Liu, Rita Nanda, Laura van ‘t Veer, I-SPY 2 Investigators. Personalized ctDNA as a predictive biomarker in high-risk early stage breast cancer (EBC) treated with neoadjuvant chemotherapy (NAC) with or without pembrolizumab (P) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-02.

Genetic Identification of all Four Mangrove Mud Crab Species (genus Scylla) Using Multiple Molecular Markers.

Misleading identification and subsequent publications on biological, molecular, and aquaculture data of mangrove mud crab (genus Scylla de Hann 1833) is a major concern in many countries. In this study, multiple molecular markers were used for genetic identification of all four known mud crab species under genus Scylla collected from India, Philippines, Myanmar, Malaysia and Indonesia. Internal Transcribed Spacer (ITS-1), Polymerase chain reaction (PCR)-Restriction Fragment Length Polymorphism (PCR-RFLP) and PCR-based species-specific markers were used to resolve taxonomic ambiguity. PCR-RFLP techniques using NlaIV and BsaJI restriction endonucleases were efficient to differentiate four different mud crab species under genus Scylla with specific fragment profile. The results also justified the use of ITS-1 and PCR-based species-specific markers to identify mud crab species available in many countries quite rapidly and effectively. Several new molecular markers generated during the study are reported here to resolve the taxonomic ambiguity of Scylla species and the results reconfirmed that India is only having two commonly available mud crab species which was reported by the authors earlier.

MeV-Stealth: A CD46-specific oncolytic measles virus resistant to neutralization by measles-immune human serum

The frequent overexpression of CD46 in malignant tumors has provided a basis to use vaccine-lineage measles virus (MeV) as an oncolytic virotherapy platform. However, widespread measles seropositivity limits the systemic deployment of oncolytic MeV for the treatment of metastatic neoplasia. Here, we report the development of MeV-Stealth, a modified vaccine MeV strain that exhibits oncolytic properties and escapes antimeasles antibodies in vivo. We engineered this virus using homologous envelope glycoproteins from the closely-related but serologically non-cross reactive canine distemper virus (CDV). By fusing a high-affinity CD46 specific single-chain antibody fragment (scFv) to the CDV-Hemagglutinin (H), ablating its tropism for human nectin-4 and modifying the CDV-Fusion (F) signal peptide we achieved efficient retargeting to CD46. A receptor binding affinity of ~20 nM was required to trigger CD46-dependent intercellular fusion at levels comparable to the original MeV H/F complex and to achieve similar antitumor efficacy in myeloma and ovarian tumor-bearing mice models. In mice passively immunized with measles-immune serum, treatment of ovarian tumors with MeV-Stealth significantly increased overall survival compared with treatment with vaccine-lineage MeV. Our results show that MeV-Stealth effectively targets and lyses CD46-expressing cancer cells in mouse models of ovarian cancer and myeloma, and evades inhibition by human measles-immune serum. MeV-Stealth could therefore represent a strong alternative to current oncolytic MeV strains for treatment of measles-immune cancer patients.

Evaluation of Analytical Performance of Seven Rapid Antigen Detection Kits for Detection of SARS-CoV-2 Virus.

BackgroundEarly diagnosis of the novel coronavirus disease of 2019 (COVID-19) in asymptomatic and symptomatic patients is crucial to identify infectious individuals and to help prevent the spread of the virus in the community. Several assays have been developed and are in use in today’s clinical practice. These assays vary in their analytical and clinical performance. For an accurate diagnosis, medical professionals must become more familiar with the test’s utility to select the most appropriate test. This study aims to evaluate the analytical performance of rapid antigen tests used for the detection of SARS-CoV-2 viral antigen compared to RT-PCR SARS-CoV-2 molecular assay.MethodsOropharyngeal swab specimens from five COVID-19 patients were tested by seven rapid antigen tests developed by different IVD companies. RT-PCR to detect specific RNA fragments of SARS-CoV-2 was used as a confirmatory test. The cycle threshold (Ct) value, which often reflects viral load, in these specimens ranged from 15 to 35. For the analytical evaluation, extraction fluid of each antigen kit was spiked with attenuated ATCC virus at different concentrations ranging from 4.6x104/mL to 7.5x105/mL and tested with antigen testing kits.ResultsOut of five confirmed positive SARS-CoV-2 specimens by RT-PCR, only one sample showed a positive result by one of the seven evaluated antigen testing kits. The positive result was observed in the specimen with a Ct value of 15. All other evaluated rapid tests were negative for all five positive specimens. This was further confirmed with the spiking study using ATCC attenuated virus, where extraction fluid of each rapid test was spiked with concentrations ranging from 4.6x104/mL to 7.5x105/mL. None of these spiked specimens showed positive results, indicating very low sensitivity of these antigen kits.ConclusionThis comparison study shows that rapid antigen tests are less sensitive than RT-PCR tests and are not reliable tests for testing asymptomatic patients, who often carry low viral load. Analytical performance of rapid antigen tests should be thoroughly evaluated before implementing it at clinical decision level.

Molecular Species Identification of Six Forensically Important Iranian Flesh Flies (Diptera).

Background:Flesh flies (Diptera: Sarcophagidae) are considered as myiasis agents and important evidences in forensic investigations. However, their use has been restricted because, at all larval stages and female adults, morphological species identification is difficult or very challenging. This study investigated to test utility of mitochondrial cytochrome oxidase subunit I (mt-COI) sequences for differentiation of six forensically important Iranian flesh flies namely, Sarcophaga crassipalpis, S. flagellifera, S. hirtipes, S. aegyptica, S. africa and S. argyrostoma.Methods:Male specimens were morphologically identified to species level and then the genomic DNA of the flies were extracted and subjected to polymerase chain reaction (PCR) against mt-COI gene. The PCR products were sequenced and the obtained sequences were analyzed for the species specific restriction fragment length polymorphisms (RFLPs).Results:Rate of genetic variation between species was 6–10% which was enough to find restriction enzymes (RE) that were able to produce species-specific RFLP profiles. Combinations of three REs: BsrFI, RsaI and HinfI, provided diagnostic bands for identification of the six Sarcophaga species.Conclusion:The results of this study showed that molecular markers such as RFLPs enhancing the use of evidence from flesh flies in forensic investigation. However, lack proper restriction sites in the COI region inhibited introduction of a single restriction enzyme for easy species identification. It is recommended to apply larger part of DNA such as combination of COI and COII genes to provide better RFLP markers for species identification of flesh flies.

Endotrophin, a pro-peptide of Type VI collagen, is a biomarker of survival in cirrhotic patients with hepatocellular carcinoma.

Aim:Type VI collagen, is emerging as a signaling collagen originating from different types of fibroblasts. A specific fragment of Type VI collagen, the pro-peptide, is also known as the hormone endotrophin. We hypothesized that this fibroblast hormone would be of particular relevance in cancer types with a high amount of fibrosis activity, namely for outcome in hepatocellular carcinoma (HCC) cirrhotic patients.Patients & methods:Plasma C6M, PRO-C6 and alphafeto-protein (AFP) were assessed in 309 patients with mixed etiologies (hepatitis C, hepatitis B, alcohol and nonalcoholic fatty liver) diagnosed as cirrhotics, cirrhotics with HCC, noncirrhotics and healthy controls. Progression-free survival and overall survival (OS) data were collected up to 6120 days after diagnosis. The ability of each marker to predict survival was investigated.Results & conclusion:The level of endotrophin assessed by PRO-C6 was able to separate healthy controls, noncirrhotics and cirrhotics from HCC (p < 0.05–0.0001). Both endotrophin and C6M provided value in the prediction of OS in cirrhotic patients with HCC. In the multivariate analysis for identifying HCC, in patients with high endotrophin (highest quartile) and that were positive for AFP (≥20 IU/ml), the hazard ratio for predicting OS was increased from 3.7 (p = 0.0006) to 14.4 (p = 0.0001) when comparing with AFP positive as a stand-alone marker. In conclusion, plasma levels for markers of Type VI collagen remodeling were associated with survival in cirrhotic patients with HCC. A combination of AFP with endotrophin improved the prognostic value compared with AFP alone for predicting OS in cirrhotic patients with HCC.

Evaluation of a real-time recombinase polymerase amplification assay for rapid detection of Schistosoma haematobium infection in resource-limited setting

Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs as well as achieving the WHO 2012–2020 road map for the total eradication of schistosomiasis. Recombinase polymerase amplification (RPA) has emerged as a rapid and simple molecular tool adaptable for fewer resources with diagnostic accuracy similar to polymerase chain reaction (PCR). This rapid molecular assay employs the use of enzymes for the amplification of nucleic acid taget at a constant temperature. The aim of this study was to validate a real-time RPA assay targeting the Dra 1 repittitive sequence of Schistosoma (S.) haematobium and evaluate its use in urogenital schistosomiasis diagnosis. S. haematobium Dra 1 molecular DNA standard was applied to determine the assay's analytical sensitivity. DNA extracts of S. haematobium, other Schistosoma species, protozoa and bacteria species were used to determine the specificity of the RPA assay. Clinical performance of the assay was validated with a panel of 135 urine samples from volunteers of schistosomiasis endemic communities. The developed assay was evaluated with urine samples extracted by just boiling and with SpeedXtract® DNA extraction kit. A specific fragment of S. haematobium Dra 1 repetitive sequence was amplified within 15 minutes at a constant 42˚C using the developed S. haematobium RPA assay. The detection limit was 15 copies of Dra1 molecular DNA standard per reaction. There was no cross-reaction with other protozoan and bacterial species except Schistosoma species, S. mansoni and S. japonicum. Using 135 urine samples, Schistosoma RPA assay had a clinical sensitivity and specificity of 98.4% (95% CI, 91.6-100) and 100% (95% CI, 94.9-99) respectively when compared to S. haematobium Dra 1 qPCR assay. The diagnostic performance of S. haematobium real-time RPA assay was not affected by the use of crude DNA extracted samples. The S. haematobium RPA assay can serve as an alternative to PCR, especially in low resource settings.

COMP (Cartilage Oligomeric Matrix Protein) Neoepitope: A Novel Biomarker to Identify Symptomatic Carotid Stenosis.

COMP (cartilage oligomeric matrix protein) is abundantly expressed in the cardiovascular system, cartilage, and atherosclerotic plaques. We investigated if the total COMP (COMPtotal) and COMP neoepitope (COMPneo) with other cardiovascular markers and clinical parameters could identify symptomatic carotid stenosis. Approach and Results: Blood samples were collected from patients with symptomatic carotid stenosis (stenosis, n=50), patients with stroke without carotid stenosis but small plaques (plaque, n=50), and control subjects (n=50). COMPtotal and COMPneo were measured using an ELISA. Ninety-two cardiovascular disease markers were measured by the Olink CVD kit. The presence of native COMP and COMPneo was determined by immunohistochemistry. The concentration of COMPneo was higher and COMPtotal was lower in the stenosis group. When the concentration was compared between the stenosis and control groups, IL-1ra (interleukin-1 receptor antagonist protein), IL6 (interleukin-6), REN (Renin), MMP1 (matrix metalloproteinase-1), TRAIL-R2 (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), ITGB1BP2 (integrin beta 1 binding protein 2), and COMPneo were predictive of stenosis. Conversely, KLK6 (kallikrein-6), COMPtotal, NEMO (nuclear factor-kappa-B essential modulator), SRC (Proto-oncogene tyrosine-protein kinase Src), SIRT2 (SIR2-like protein), CD40 (cluster of differentiation 40), TF (tissue factor), MP (myoglobin), and RAGE (receptor for advanced glycation end-products) were predictive of the control group. Model reproducibility was good with the receiver operating characteristic plot area under the curve being 0.86. When comparing the plaque group and stenosis group, COMPneo, GAL (galanin), and PTX3 (pentraxin-related protein PTX3) were predictive of stenosis. Model reproducibility was excellent (receiver operating characteristic plot area under the curve 0.92). COMPneo was detected in smooth muscle-, endothelial-, and foam-cells in carotid stenosis. Degradation of COMP may be associated with atherosclerosis progression and generation of a specific COMP fragment-COMPneo. This may represent a novel biomarker that together with COMPtotal and other risk-markers could be used to identify symptomatic carotid stenosis. Graphic Abstract: A graphic abstract is available for this article.

Improved metabolite characterization by liquid chromatography – Tandem mass spectrometry through electron impact type fragments from adduct ions

Using a chimeric collision cell mounted on a quadrupole time-of-flight platform, collision induced dissociation (CID) and electron induced dissociation (EID) were investigated for the LC-MS analysis of low molecular weight compounds including drugs and endogenous metabolites. Compared to CID, EID fragmentation of the [M+H]+ species (10–20 eV) from standard compounds resulted in additional specific and informative fragments, mostly due to neutral losses and, in some cases due to ring openings. Some analytes, for example reserpine and vinpocetine, provided characteristic [M+H]•2+ species. For most analytes for sodium and potassium adducts and multimers a radical cation M•+ and electron impact type fragments were observed in the EID spectra, providing the opportunity to use EI libraries to support metabolite identification. EID opens the possibility to get structural information from adduct ions which is often not the case with CID. EID enabled the putative characterization of two metabolites in rat urine as glucuronides of 5,6-dihydroxyindole based on EID fragmentation of the potassium adducts.

Structural variation and phylogenetic relationship of Geospiza magnirostris based on mitochondrial control region

The control region of mitochondrial genome is the main non-coding region. There is no details about Geospiza magnirostris’s control region, who is the only species of Geospiza in Darwin finches whose mitochondrial genome has been sequenced. In this study, the control region mitochondrial genome of G. magnirostris was analyzed. Compare research for 15 Passerine species, belonging to 4 families (Darwin ground finch, Thraupidae, Icteridae, Emberizidae), was also been done. The result showed the length was 1202 bp and it could been divided into three parts, domainI, domainII and domainIII. Genetic distances showed average divergence of different family ranged from 0.36% (Darwin ground finch) to 9.20% (Emberizidae), indicated that the Darwin ground finch may be a very conservative single-source species. Some specific sequence fragments such as conservative sequence box, boxes C, D, F and E, HSP/LSP, CSB-1, TAS-1 were also found in the control region of G. magnirostris, while B-box, CSB-2 and CSB-3 were not found in it. The F-box was detected in all selected 15 species and was 29 bp in length with no variant sites. In the E-box, 20 of the 21 nucleotide positions were identical as conserved among the selected species. There are two variant sites in the D-box, and five variant sites in the C-box. Basing the phylogenetic tree, it can be clearly seen that the relationship of G. magnirostris and family Thraupidae is the closest in all selected family. Through this study, the structure characteristics of G. magnirostris control region were explored and understood. This study provided more basic information for in-depth research of G. magnirostris. It’s also reminded us that we should pay more attention to explore the characteristics of the control region of Darwin’s finches.

Exploring DNA Diversity in Leathers:

DNA based approaches have become widespread in recent times to identify the origin of samples when its phenotypic characteristics are not distinguishable. This, in particular, applies to the leather industry wherein with an increase in duplicated embossing of grain patterns; there is a need to detect the animal origin of commercial leather articles. Thus, the characterization of molecular markers that enables rapid detection of the leather source helps us in precise species identification. The present study aims to generate definite sequences between the four major species in the Bovidae family (Buffalo, Cow, Goat, and Sheep), which are the major players in the manufacture of leather products, especially in India. Based on specific mitochondrial sequences, a specific fragment of the mitochondrial 12SrRNA gene was amplified by PCR as a marker for species-level identification. By the maximum homogeneity, from the NCBI and BOLD database, the BLAST analysis of the sequences of amplicons from unknown sources, distinguish closely related species of the subfamilies Bovinae (buffalo and Cow) and Caprinae (sheep and goat) and this 12SrRNA based PCR-BLAST analysis is a good tool to identify the origin and control the quality of leathers that are being manufactured. The present study has optimized an approach for the extraction and amplification of DNA from the finished leather, which is one of the most significant challenges because of the vigorous processes encountered during their manufacture. The findings of the study have commercial value at large scale.

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE.

Genome editing technologies include techniques used for desired genetic modifications and allow the insertion, modification or deletion of specific DNA fragments. Recent advances in genome biology offer unprecedented promise for interdisciplinary collaboration and applications in gene editing. New genome editing technologies enable specific and efficient genome modifications. The sources that inspire these modifications and already exist in the genome are DNA degradation enzymes and DNA repair pathways. Six of these recent technologies are the clustered regularly interspaced short palindromic repeats (CRISPR), leveraging endogenous ADAR for programmable editing of RNA (LEAPER), recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing (RESTORE), chemistry-based artificial restriction DNA cutter (ARCUT), single homology arm donor mediated intron-targeting integration (SATI), RNA editing for specific C-to-U exchange (RESCUE). These technologies are widely used from various biomedical researches to clinics, agriculture, and allow you to rearrange genomic sequences, create cell lines and animal models to solve human diseases. This review emphasizes the characteristics, superiority, limitations, also whether each technology can be used in different biological systems and the potential application of these systems in the treatment of several human diseases.

The influence of author’s provocation upon interpretation of the fourth chapter of “Finnegans Wake” by James Joyce

This article is dedicated to the peculiarities of the last novel of the Irish writer James Joyce &amp;ldquo;Finnegans Wake&amp;rdquo;, written in 1939. James Joyce paid deliberate attention to the linguistic arrangement of his work, resulting in the novel becoming difficult to translate, as well as to read and comprehend. Analysis is conducted on the fourth chapter of &amp;ldquo;Finnegans Wake&amp;rdquo; for demonstrating a peculiar feature of James Joyce's style of writing. Provocation of the Irish novelist is consists in usage of various puns for confusing the reader. This instigates the reader to seek different meanings that correspond to the writer&amp;rsquo;s concept throughout the text or a specific fragment. The article employs semantic and structural methods of analysis for interpretation of pun. The research also uses historical- cultural and biographical methods for analyzing the complex instances of interpretation. Field analysis is applied for allocating the acquired results into three zones: nuclear, close, and far peripheries. The main result of this research consists in analysis of the novel in the context of author's provocation. The fancy linguistic arrangement of the literary text should be viewed from the two perspectives: on the one hand, the reader analyzes the language and perceives pun as an intended concealment of the novel&amp;rsquo;s message; on the other hand, interrelation of the meanings of one pun with forces the reader to ponder on a play of sorts between the author and the reader. It can be unequivocally claimed that the reader is constantly uncertain in their correctness.