An activatable chemiluminescence probe based on phenoxy-dioxetane scaffold for biothiol imaging in living systems

Quantification of biothiols in living systems is essential to understand their biological applications. Here, we developed two activatable chemiluminescence probes (SHCL and NCCL) and investigated their utility in the bioimaging of intracellular biothiols by directly tethering 2,4-dinitrobenzenesulfonyl to the hydroxyl group of phenoxy-dioxetane. The design of these two probes differed in substituents of phenol-dioxetane, i.e., SHCL contained the ortho chlorine, whereas NCCL had the para hydroxymethyl. Upon glutathione (GSH) cleavage, both probes emitted significantly “turn-on” chemiluminescent signals. However, the chemiluminescence intensity based on NCCL declined with increasing GSH level above 5 mM, while SHCL exhibited much higher chemiluminescent intensity and a wider concentration range (0.5 μM-50 mM), which was much more suitable for sensing endogenous biothiols.
We further demonstrated that chlorine substitution in SHCL played an important role in bioimaging owing to the halogen effect, providing a lower pKa value and significant enhancement of the chemiluminescent emission. SHCL imaged the biothiols effectively in tumor cells and tumor-bearing mice. Additionally, this novel chemiluminescence probe can be easily used to evaluate the in vitro activity of acetylcholinesterase. Overall, we anticipate that SHCL may provide a facile and intuitive tool for studying the role of biothiols in diseases.

Nitrogen doped graphene quantum dots based long-persistent chemiluminescence system for ascorbic acid imaging.

  • High photo-intensity and sluggish flight attenuation are important to highly sensitive chemluminescence imaging. Herein, we present a copper ion catalyzed long-persistent chemiluminescent imaging system of nitrogen-doped graphene quantum dots (NGQDs) for ascorbic acid detection in fruit. NGQDs as luminescent probe are fabricated, emitting out chemluminescence with the direct oxidation by H2O2.
  • In addition, Cu2+ ion enlarges over two order magnitudes of NGQDs CL intensity (214 times) due to its catalyzed Fenton-like reaction for H2O2 decomposition, and displaying unique specificity against other metal ions. As a result, the twinkling luminescence of NGQDs is boosted and changes to hold persistent with small decay in the presence of copper ion exhibiting potential for CL imaging.
  • As an imaging model, a visual sensor based on Cu2+/NGQDs/H2O2 is developed for AA quantitative monitoring with a limit of detection (LOD) of 0.5μM (S/N=3) and applied in real AA detection in fruit. The CL imaging method demonstrated with high stability and proper sensitivity would provide a convenient and visual tool for AA determination, displaying promising candidates for imaging sensing.

Imaging systems for westerns: chemiluminescence vs. infrared detection.

Western blot detection methods have traditionally used X-ray films to capture chemiluminescence. The increasing costs for film, reagents, and maintenance have driven researchers away from darkrooms to more sensitive and technologically advanced digital imaging systems. Cooled charge coupled devices (CCD) cameras capture both chemiluminescence and fluorescence images, with limitations for each detection method. Chemiluminescence detection is highly sensitive and relies on an enzymatic reaction that produces light, which can be detected by a CCD camera that records photons and displays an image based on the amount of light generated. However, the enzymatic reaction is dynamic and changes over time making it necessary to optimize reaction times and imaging.
Fluorescent detection with a CCD camera offers a solution to this problem since the signal generated by the proteins on the membrane is measured in a static state. Despite this advantage, many researchers continue to use chemiluminescent detection methods due to the generally poor performance of fluorophores in the visible spectrum. Infrared imaging systems offer a solution to the dynamic reactions of chemiluminescence and the poor performance of fluorophores detected in the visible spectrum by imaging fluorphores in the infrared spectrum.
Infrared imaging is equally sensitive to chemiluminescence and more sensitive to visible fluorescence due in part to reduced autofluorescence in the longer infrared wavelength. Furthermore, infrared detection is static, which allows a wider linear detection range than chemiluminescence without a loss of signal.
A distinct advantage of infrared imaging is the ability to simultaneously detect proteins on the same blot, which minimizes the need for stripping and reprobing leading to an increase in detection efficiency. Here, we describe the methodology for chemiluminescent (UVP BioChemi) and infrared (LI-COR Odyssey) imaging, and briefly discuss their advantages and disadvantages.

Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots.

A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown.
The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence. The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 x 10(-18) mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images.

New advanced oxidation progress with chemiluminescence behavior based on NaClO triggered by WS 2 nanosheets

As one integral part of coping strategies for addressing water pollution, advanced oxidation progresses (AOPs) get enormous attentions in recent years. However, the complex synthesis and high cost of H2O2 and K2S2O8 hampered their developments. Herein, a novel AOP with the chemiluminescence (CL) property based on economic NaClO and WS2 nanosheets was proposed to achieve efficient decomposition of organic pollutants.
In this AOP, WS2 nanosheets exhibited a dual-function feature of the catalyst and energy acceptor. It demonstrated that the reaction order of WS2 nanosheets was equal to 0.8271 and enormous singlet oxygen (1O2),·ClO and hydroxyl radical (·OH) were generated in rhodamine B (RhB) degradation process. Interestingly, a strong CL emission was observed and reflected the relative concentration of 1O2 and·OH for adjusting the oxidizing capability in WS2 nanosheets-NaClO system.
Through a series of degradation tests, RhB, methylene blue (MB), p-nitrophenol and phenol were decomposed and the degradation efficiency of over 90% was achieved. Therefore, this study not only builds a chemiluminescent AOPs to eliminate organic pollutants, but also broadens the applications of WS2 nanomaterials and CL in environmental field.

ATP Chemiluminescence Assay Kit

E-BC-F002-48T Elabscience Biotech 48T 3 EUR

ATP Chemiluminescence Assay Kit

E-BC-F002-96T Elabscience Biotech 96T 450 EUR

ATP Chemiluminescence Assay Kit

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ATP Chemiluminescence Assay Kit

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ATP Chemiluminescence Assay Kit

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ATP Chemiluminescence Assay Kit

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ATP Chemiluminescence Assay Kit

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ATP Chemiluminescence Assay Kit

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SuperPico ECL Chemiluminescence Kit

E422-00-10ml Vazyme 10 ml 1.09 EUR

SuperPico ECL Chemiluminescence Kit

E422-01-100ml Vazyme 100 ml 51.23 EUR

SuperPico ECL Chemiluminescence Kit

E422-02-500ml Vazyme 500 ml 210.37 EUR

SuperFemto ECL Chemiluminescence Kit

E423-01 Vazyme 100ml 168.5 EUR

SuperFemto ECL Chemiluminescence Kit

E423-02 Vazyme 500ml 714 EUR

SuperFemto ECL Chemiluminescence Kit

E423-00-10ml Vazyme 10 ml 1.09 EUR

SuperFemto ECL Chemiluminescence Kit

E423-01-100ml Vazyme 100 ml 174.4 EUR

SuperFemto ECL Chemiluminescence Kit

E423-02-500ml Vazyme 500 ml 739.57 EUR

Automatic Chemiluminescence Immunoassay Analyzer

ACIR-100 Hangzhou AllTest Biotech 1 kit Ask for price

Automatic Chemiluminescence Immunoassay Analyzer

ACIR-200 Hangzhou AllTest Biotech 1 kit Ask for price

Total Proinsulin Chemiluminescence Kit (5 Kit Pack)

90112 Crystal Chem 96x5 2340 EUR

Intact Proinsulin Chemiluminescence Kit (5 Kit Pack)

90107 Crystal Chem 96x5 2340 EUR

Highly Tough, Stretchable, and Solvent-Resistant Cellulose Nanocrystal Photonic Films for Mechanochromism and Actuator Properties

Cellulose nanocrystals (CNCs)-derived photonic materials have confirmed great potential in producing renewable optical and engineering areas. However, it remains challenging to simultaneously possess toughness, strength, and multiple responses for developing high-performance sensors, intelligent coatings, flexible textiles, and multifunctional devices. Herein, the authors report a facile and robust strategy that poly(ethylene glycol) dimethacrylate (PEGDMA) can be converged into the chiral nematic structure of CNCs by ultraviolet-triggered free radical polymerization in an N,N-dimethylformamide solvent system.
The resulting CNC-poly(PEGDMA) composite exhibits impressive strength (42 MPa), stretchability (104%), toughness (31 MJ m-3 ), and solvent resistance. Notably, it preserves vivid optical iridescence, displaying stretchable variation from red, yellow, to green responding to the applied mechanical stimuli. More interestingly, upon exposure to spraying moisture, it executes sensitive actuation (4.6° s-1 ) and multiple complex 3D deformation behaviors, accompanied by synergistic iridescent appearances.
Due to its structural anisotropy of CNC with typical left-handedness, the actuation shows the capability to generate a high probability (63%) of right-handed helical shapes, mimicking a coiled tendril. The authors envision that this versatile system with sustainability, robustness, mechanochromism, and specific actuating ability will open a sustainable avenue in mechanical sensors, stretchable optics, intelligent actuators, and soft robots.

Understanding the Drying Behavior of Regenerated Cellulose Gel Beads: The Effects of Concentration and Nonsolvents

The drying behavior of regenerated cellulose gel beads swollen with different nonsolvents (e.g., water, ethanol, water/ethanol mixtures) is studied in situ on the macroscopic scale with an optical microscope as well as on nanoscale using small-angle/wide-angle X-ray scattering (SAXS/WAXS) techniques. Depending on the cellulose concentration, the structural evolution of beads during drying follows one of three distinct regimes.
First, when the cellulose concentration is lower than 0.5 wt %, the drying process comprises three steps and, regardless of the water/ethanol mixture composition, a sharp structural transition corresponding to the formation of a cellulose II crystalline structure is observed. Second, when the cellulose concentration is higher than 5.0 wt %, a two-step drying process is observed and no structural transition occurs for any of the beads studied. Third, when the cellulose concentration is between 0.5 and 5.0 wt %, the drying process is dependent on the nonsolvent composition.
A three-step drying process takes place for beads swollen with water/ethanol mixtures with a water content higher than 20%, while a two-step drying process is observed when the water content is lower than 20%. To describe the drying behavior governed by the cellulose concentration and nonsolvent composition, a simplified phase diagram is proposed.

Cellular Flocculation Using Concentrated Polymer Brush-Modified Cellulose Nanofibers with Different Fiber Lengths

In this study, concentrated polymer brush-modified cellulose nanofibers (CNFs) with different fiber lengths were used for the flocculation of cells for systematically studying the mechanism of this unique cellular flocculation based on colloidal flocculation theory. Concentrated poly(p-styrenesulfonic acid sodium salt) brush-grafted CNF (CNF-PSSNa) with different fiber lengths were cultured with three different cell types to examine their influence on floc (cell clusters formed by cellular flocculation) characteristics. The floc size and survival rate could be controlled by modifying the CNF-PSSNa fiber lengths.
The three cell types showed the same flocculation tendency after culture, indicating the applicability of the method in different cell lines. After 2 weeks of culture, CNF-PSSNa increased the specific expression of hepatocytes compared to the two-dimensional cell culture. Thus, owing to its wide applicability, high cell viability, and ability to control cell size and improve cell function, this technology could be used as a new three-dimensional cell culture method.

Gold nanoparticles spontaneously grown on cellulose nanofibrils as a reusable nanozyme for colorimetric detection of cholesterol in human serum

Recently, gold nanoparticles (AuNPs) are extensively used as peroxidase mimics. However, low catalytic activity, high synthesis cost, substrate-induced aggregation in reaction medium and difficulty in recovery and reuse still remain as major challenges. Here, a novel, simple, spontaneous, and reagent-less in-situ method for the production of AuNPs using dialdehyde cellulose nanofibrils (DACNF) is proposed. AuNPs synthesis time and size were greatly influenced by aldehyde content and the optimal aldehyde content for ultra-small AuNPs (≈10 nm) was 2.1 mM/g. AuNPs@DACNFs exhibited broad-spectrum peroxidase activity and steady-state kinetics revealed their better kinetic parameters (low Km and high Vmax) over horseradish peroxidase (HRP). AuNPs@DACNFs was further converted into paper strip, which served as a biosensor for H2O2 and cholesterol detection.
The proposed method exhibited wide linear response in the range of 10-90 μM and 0.05-0.45 mM, and detection limit of 0.39 μM and 1.9 μM for H2O2 and cholesterol, respectively. Great shelf life and reusability were evident by FE-SEM and ICP-OES analysis. The smartphone application “Color Grab” was used to enable the portable onsite detection. The results of cholesterol detection in human serum samples were in agreement with clinically observed values, suggesting the great potential of the probe in disease diagnosis.

COBL9 and COBL7 synergistically regulate root hair tip growth via controlling apical cellulose deposition

Root hairs are cylindrical extensions of root epidermal cells that are important for the acquisition of water and minerals, interactions between plant and microbes. The deposition of cell wall materials in the tip enables root hairs to maintain elongation constantly. To date, our knowledge of the regulators that connect the architecture of cell wall and the root hair development remains very limited.
Here, we demonstrated that COBL9 and COBL7, two genes of COBRA-Like family in Arabidopsis as well as their counterparts in rice, OsBC1L1 and OsBC1L8, regulate root hair growth. Single mutant cobl9, double mutants cobl7 cobl9 and double mutants osbc1l1 osbc1l8 all displayed prematurely terminated root hair elongation, though at varying levels. COBL7-YFP and COBL9-YFP accumulate prominently in the growing tips of newly emerged root hairs.
Furthermore, cobl9, cobl7 cobl9 and osbc1l1 osbc1l8 mutants were defective in the enrichment of cellulose in the tips of the growing root hairs. We also discovered that overexpression of COBL9 could promote root hair elongation and salinity tolerance. Taken together, these results provide compelling evidence that the polarized COBL7 and COBL9 in the tip of the emerging root hairs have conserved roles in regulating root hair development and stress adaptation in dicots and monocots.

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Efficacy and safety of endovascular brachytherapy combined with transarterial chemoembolization for the treatment of hepatocellular carcinoma patients with type III or IV portal vein tumor thrombosis

Background: The purpose of this study was to evaluate the efficacy and safety of endovascular brachytherapy (EVBT) combined with transarterial chemoembolization (TACE) for the treatment of hepatocellular carcinoma (HCC) complicated with type III OR IV portal vein tumor thrombosis (PVTT) and to further analyze the prognostic predictors for the patients with HCC and PVTT.
Methods: We retrospectively analyzed the medical records of 54 patients who were diagnosed with HCC complicated with type III or IV PVTT and received EVBT combined with modified TACE treatment from January 2017 to June 2019. Adverse events, treatment response, overall survival (OS), progression-free survival (PFS), and stent patency were analysed to evaluate the efficacy and safety of this treatment. The independent prognostic predictors of OS were also statistically analyzed by the cox regression model.
Results: No adverse events occurred in the enrolled patients receiving EVBT combined with TACE treatment. The objective response and disease control rates were 42.6% and 96.3% respectively within 4 weeks after the treatment. The median OS and PFS were 209 days and 138 days, respectively. Cumulative stent patency rate was 70.4% at the last follow-up. AFP ≥ 400 ng/ml, ECOG PS > 1, Child Pugh grade B, and non-hemihepatic HCC were independent risk predictors to evaluate the OS of HCC patient with type III or IV PVTT.
Conclusions: EVBT combined with TACE was a relatively effective and safe strategy to treat HCC patients with type III or IV PVTT.

Dual immune checkpoint blockade in hepatocellular carcinoma: where do we stand?

Hepatocellular carcinoma (HCC) represents the fourth most common cause of cancer-related death. Surgery, local ablative therapies and liver transplantation are the only potentially curative strategies, but the majority of patients present with advanced disease at diagnosis or develop recurrence after surgery.
In recent years, immunotherapy for HCC has received growing interest, and one of the most promising strategies is the association of two immune checkpoint inhibitors (ICIs), which has already demonstrated its potential in other solid tumors such as melanoma and renal cell carcinoma. Herein, we discuss the role and the biologic rationale of dual immune checkpoint blockade in HCC patients, focusing on the two ICI combinations: nivolumab plus ipilimumab and durvalumab plus tremelimumab.

Exposome and Skin. Part 2. The Influential Role of the Exposome, Beyond UVR, in Actinic Keratosis, Bowen’s Disease and Squamous Cell Carcinoma: A Proposal

Actinic keratosis (AK) is the main risk factor for the development of cutaneous invasive squamous cell carcinoma (SCC). It represents the first sign of severe chronic ultraviolet radiation exposure, which has a clear significant effect. Nevertheless, the skin is exposed to many other exposome factors which should be thoroughly considered. Our aim was to assess the impact of exposome factors other than ultraviolet radiation (UVR) on the etiopathology of AK and Bowen’s disease (BD) and progression of AK to SCC and to design tailored prevention strategies.
We performed an exhaustive literature search in September 2021 through PubMed on the impact of exposome factors other than UVR on AK, BD and SCC. We conducted several parallel searches combining terms of the following topics: AK, BD, SCC and microbiome, hormones, nutrition, alcohol, tobacco, viral infections, chemical contaminants and air pollution. Notably, skin microbiome studies have shown how Staphylococcus aureus infections are associated with AK and AK-to-SCC progression by the production of chronic inflammation. Nutritional studies have demonstrated how a caloric restriction in fat intake, oral nicotinamide and moderate consumption of wine significantly reduce the number of premalignant keratoses and SCC.
Regarding lifestyle factors, both alcohol and smoking are associated with the development of SCC in a dose-dependent manner. Relevant environmental factors are viral infections and chemical contaminants. Human papillomavirus infections induce deregulation of cellular proliferation and are associated with AK, BD and SCC. In addition to outdoor jobs, occupations such as industrial processing and farming also increase the risk of developing keratoses and SCC. The exposome of AK will undoubtedly help the understanding of its etiopathology and possible progression to SCC and will serve as a basis to design tailored prevention strategies.

A Genome-Wide Investigation of Effects of Aberrant DNA Methylation on the Usage of Alternative Promoters in Hepatocellular Carcinoma

Background: The alternative usage of promoters provides a way to regulate gene expression, has a significant influence on the transcriptome, and contributes to the cellular transformation of cancer. However, the function of alternative promoters (APs) in hepatocellular carcinoma (HCC) has not been systematically studied yet. In addition, the potential mechanism of regulation to the usage of APs remains unclear. DNA methylation, one of the most aberrant epigenetic modifications in cancers, is known to regulate transcriptional activity. Whether DNA methylation regulates the usage of APs needs to be explored. Here, we aim to investigate the effects of DNA methylation on usage of APs in HCC.
Methods: Promoter activities were calculated based on RNA-seq data. Functional enrichment analysis was implemented to conduct GO terms. Correlation tests were used to detect the correlation between promoter activity and methylation status. The LASSO regression model was used to generate a diagnostic model. Kaplan-Meier analysis was used to compare the overall survival between high and low methylation groups. RNA-seq and whole-genome bisulfite sequencing (WGBS) in HCC samples were performed to validate the correlation of promoter activity and methylation.
Results: We identified 855 APs in total, which could be well used to distinguish cancer from normal samples. The correlation of promoter activity and DNA methylation in APs was observed, and the APs with negative correlation were defined as methylation-regulated APs (mrAPs). Six mrAPs were identified to generate a diagnostic model with good performance (AUC = 0.97). Notably, the majority of mrAPs had CpG sites that could be used to predict clinical outcomes by methylation status. Finally, we verified 85.6% of promoter activity variation and 92.3% of methylation changes in our paired RNA-seq and WGBS samples, respectively. The negative correlation between promoter activity and methylation status was further confirmed in our HCC samples.
Conclusion: The aberrant methylation status plays a critical role in the precision usage of APs in HCC, which sheds light on the mechanism of cancer development and provides a new insight into cancer screening and treatment.

A case of locally advanced adenosquamous carcinoma of the cecum with long-term survival

A 63-year-old woman was admitted to our hospital with a right lower abdominal mass and general fatigue. Preoperative examination suggested a large ovarian tumor or cecal carcinoma. However, her intraoperative diagnosis was colon cancer; we therefore performed an ileocecal resection with oophorectomy. The tumor was pathologically diagnosed as adenosquamous carcinoma T4bN1M-stage IIIa.
We administrated CapeOX adjuvant chemotherapy for 6 months. Adenosquamous carcinoma is extremely rare, at around 0.1% of all colorectal cancers, and usually has a poor prognosis. The patient is still alive without recurrence after 84 post-operative months, even with later developments of metachronous early colorectal cancer and breast cancer. We herein report a rare case of cecal ASC with good prognosis.

Whatman FTA cards versus plasma specimens for the quantitation of HIV-1 RNA using two real-time PCR assays

Background: Several studies have compared the use of dried blot spot (DBS) as an alternative to plasma specimens, mainly using Whatman 903 cards as filter paper. The aim of this study was to evaluate the use of Whatman FTA card (FTA card) specimens for HIV-1 viral load testing compared to plasma specimens using two real-time PCR assays manufactured by Roche and Abbott.
Methodology: A cross-sectional study was conducted between April 2017 and September 2017 on HIV-1 patients admitted to Yalgado Ouédraogo Teaching Hospital. Paired FTA cards and plasma specimens were collected and analysed using the Abbott Real-Time HIV-1 assay (Abbott) and COBAS AmpliPrep/COBAS TaqMan v2.0 (Roche).
Results: In total, 107 patients were included. No statistical differences (P>0.05) were observed between the mean viral loads obtained from the FTA cards and those of the plasma specimens using the Roche and Abbott assays. In total, 29 samples with Roche and 15 samples with Abbott assay showed discrepant results. At viral loads of ≤1000 copies ml-1, the sensitivity and specificity of the FTA cards were 78.6 and 100% with Roche, and 92.3 and 95.9% with Abbott, respectively. Both the Roche and Abbott assays showed good correlation and agreement between the FTA cards and plasma values.
Conclusion: Our study demonstrates the feasibility of using FTA card filter paper for HIV-1 viral load testing. However, further studies will be required for the validation of the use of FTA card filter paper in HIV-1 treatment monitoring.
Stability of Human Immunodeficiency Virus Serological Markers in Samples Collected as HemaSpot and Whatman 903 Dried Blood Spots.
  • Dried blood spots (DBS) are frequently used in clinical testing for biosurveillance, infectious disease and confirmatory testing, and clinical trials, particularly for populations in remote areas. The HemaSpot-HF blood collection device (HS) provides an alternative format to the Whatman 903 cards (903) to simplify sample collection and processing. In this study, the performance of the HS was compared to that of the 903 using previously characterized clinical specimens and HIV seroconversion panels known to exhibit markers of early human immunodeficiency virus (HIV) infection.
  • HS and 903 samples were prepared and tested by Bio-Rad GS HIV Combo Ag/Ab enzyme immunoassay (EIA), GS HIV-1/-2 Plus O EIA, GS HIV-1 Western blot, and HIV-1 Geenius assays. Both HS and 903 performed well for up to 6 months at room temperature, but a marked loss of Western blot and low titer antibody signals from early infection samples was observed in samples stored for 180 days at elevated (37 to 45°C) temperatures and high humidity (95%).
  • HemaSpot samples placed in sealed bags with additional desiccant were protected from degradation and showed improved signal recovery relative to that of the 903. HS was easier to use than the 903 and showed higher sensitivity and reproducibility for early infection samples and improved stability.

Whatman Protein Saver Cards for Storage and Detection of Parasitic Enteropathogens.

Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards.
DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction.
For E. histolytica, this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium, this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.

Comparison of stool collection and storage on Whatman FTA Elute cards versus frozen stool for enteropathogen detection using the TaqMan Array Card PCR assay.

The use of Polymerase Chain Reaction (PCR) assays for pathogen detection in travelers’ diarrhea (TD) field studies is limited by the on-site processing and storage requirements for fecal specimens. The objectives of this investigation were to i) characterize the pathogen distribution in deployed military personnel with TD using the TaqMan® Array Card PCR (TAC) on frozen stool and diarrheal smears on Whatman FTA Elute cards (FTA cards), and to ii) compare TAC detection of enteropathogen targets using smeared FTA cards and frozen stool, using TAC on frozen stool as the ‘reference standard’.
Stool samples, obtained from active duty personnel with acute TD enrolled in a field trial, were smeared onto FTA cards and stored at room temperature. A corresponding aliquot of stool was frozen in a cryovial. FTA cards and frozen stool samples were tested at a central lab, using a customized TAC for detection of TD pathogens. 187 paired frozen stool samples and smeared FTA cards were stored for a median of 712 days (IQR 396-750) before testing. Overall detection rates were 78.6% for frozen stool and 73.2% for FTA cards. Diarrheagenic Escherichia coli were the most common bacteria identified. Using the TAC results on frozen stool as the reference, the overall sensitivity and specificity of TAC on FTA cards was 72.9% and 98.0% respectively.
TAC on FTA cards demonstrated a decrease in sensitivity with increasing frozen stool quantification cycle (Cq) (90.0% in FTA cards with a corresponding frozen stool Cq < 30, and 72.9% in samples with a corresponding frozen stool Cq < 35). Our findings support the use and further development of FTA cards in combination with a quantitative PCR assay for enteropathogen detection in TD field studies.

Detection of anti-hepatitis C virus and hepatitis C virus RNA in dried blood spot specimens using Whatman No. 1 filter paper.

Dried blood spot (DBS) specimen simplifies blood collection, processing, storage and shipment and may reduce the cost of testing for hepatitis C virus (HCV) infection. We wanted to see if DBS using a cheap filter paper is reliable alternative to serum for detection of anti-HCV and HCV RNA.
At a tertiary care hospital in Northeast India, we collected 91 paired DBS and serum specimens from patients at risk of HCV infection from July 2014 to June 2015. DBS was collected on Whatman No. 1 filter paper. After processing, the specimens were subjected to anti-HCV detection by a third-generation Enzyme-Linked Immunosorbent Assay (ELISA). The reactive DBS and serum specimens were further subjected to HCV RNA detection by polymerase chain reaction. The results were analysed in paired screen-positive study design.
Anti-HCV was detected in 9 (9.9%) DBS specimens and 10 (10.9%) serum specimens. There was statistically significant (P < 0.0001) correlation between the optical density values of DBS and serum specimens (Pearson r = 0.9181, 95% confidence interval: 0.8781-0.9453). HCV RNA was detected in 5/9 (55.6%) reactive DBS and 9/10 (90.0%) reactive serum specimens. There was no correlation between HCV RNA levels in the DBS and the serum specimens. The relative sensitivity rate and the relative false-positive rate of DBS anti-HCV ELISA were 0.89 and 1.00, respectively.
DBS using Whatman No. 1 filter paper is quite reliable as serum for detection of anti-HCV. It can be useful in effective surveillance. However, it is not suitable for confirmation of chronic HCV infection.

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Synthesis and types of RNA

RNAs are molecules that play several fundamental roles for the cell and there are many types: the mRNAs carry DNA information outside the nucleus; tRNAs transport amino acids, the “building blocks” that make up proteins; rRNAs form ribosomes. There are also other RNAs that act as enzymes and RNAs can regulate the expression of genes.

Synthesis of RNA

It occurs in the same way that DNA is synthesized:

the helicase has the task of unrolling
the RNA polymerase has the task of synthesizing (no primer is needed, but flows the DNA strand until it finds a promoter: a sequence of nucleotides in the DNA that directly indicates where the synthesis should start) and is supported by general factors of transcription that aggregate to the promoter and place the polymerase where there is the TATA sequence (15 nucleotides upstream of the transcription start site) and start the whole
Nitrogen bases are added in the form of triphosphate nucleosides (ATP, CTP, GTP, UTP)
The synthesis continues until the RNA polymerase encounters a termination signal
The RNA detaches and the DNA spirals up again.
There are three types of RNA polymerase:

polymerase 1: tRNA, rRNA, miRNA
polymerase 2: mRNA
polymerase 3: tRNA, rRNA, miRNA
This also summarizes the other types of RNA (ribosomal, messenger and transfer).

An RNA is composed of introns and exons that must be eliminated and are located at the ends of the intron. The exons are eliminated with a process called splicing, operated by molecules of (nuclear RNA) and not by proteins. Splicing allows you to code proteins other than the same gene.

The mRNAs

MRNA is the complementary copy of the gene it transcribed. It allows the cell to amplify its synthesis activity. A DNA molecule contains information for numerous mRNA molecules. Each mRNA molecule can be translated into numerous polypeptide chains.

The mRNAs leave the nucleus to be translated into a protein thanks to the genetic code. The messenger RNA nucleotide sequences are read in triplets (codons) and transformed into amino acids. Since there are four nucleotides, 4x4x4 = 64 combinations of three nucleotides. However, there are only 20 amino acids, so an amino acid can correspond to several triplets.


All mature tRNA molecules have:

73-93 nucleotides
Traits in which the pairing of the bases makes the structure fold
Loops not paired because in those areas there are 10 unusual bases modified post-transcriptionally and therefore it is impossible to pair them
An anticodone
A binding site for amino acids (CCA) at the 3 ‘end
TRNA is used to bind amino acids together. There are 10 different aminoacyl-tRNA synthetase enzymes that bind amino acids and tRNA with an ester bond. They are fundamental for the translation of messenger RNA because they recognize and bind the codon of mRNA (thanks to their complementary anticodon) on one side and on the other the amino acid.

Some tRNAs can mate to more than one codon because they can tolerate an oscillating pair in third position. The aminoacyl-tRNA synthetase enzymes combine all the amino acids with their corresponding RNA tranfer.

The rRNAs

It represents the most stable form of RNA and is 70-80% of the total RNA. The genes that encode rRNA are highly repeated. Ribosomes are made up of rRNA and proteins. They provide support for protein synthesis because they catalyze the link between two amino acids, that is, they transform the language of mRNA to that of amino acids according to the genetic code.

Ribosomes are made up of:

Major subunit: RNA 28S, RNA 5.8S and RNA 5S and about 45 proteins
Minor subunit: RNA 18S and 33 proteins
The RNA 28S, 18S and 5.8S are derived from a single transcript cut from nucleases in rRNA 18S and 32S (28S and 5.8S). The 5S RNA comes from a different precursor RNA, synthesized outside the nucleolus and then transferred to the nucleolus.

Ribosomes have a binding site for the mRNA and tRNA molecule (site A, P and E). The amino acid-bound tRNAs are positioned at site A. The amino acid is bound to what is found at site P. The ribosome then slips and the now amino-free tRNA is found at site E waiting to be expelled.

A microfluidic DNA library preparation platform for next-generation sequencing

A microfluidic DNA library preparation platform for next-generation sequencing

Next-generation sequencing (NGS) is emerging as a strong device for elucidating genetic info for a large range of functions. Unfortunately, the surging recognition of NGS has not but been accompanied by an enchancment in automated strategies for preparing formatted sequencinglibraries.

To tackle this challenge, we have now developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for evaluation by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) pattern handling with peripheral modules to create a fully-integrated, sample-in library-out platform.

A microfluidic DNA library preparation platform for next-generation sequencing
A microfluidic DNA library preparation platform for next-generation sequencing

In this report, we use our automatedsystem to arrange NGSlibraries from samples of human and bacterial genomic DNA. E. coli libraries ready on-device from 5 ng of whole DNA yielded glorious sequence protection over the complete bacterial genome, with>>99% alignment to the reference genome, even genome protection, and good high quality scores.

Furthermore, we produced a de novo meeting on a beforehand unsequenced multi-drug resistant Klebsiella pneumoniae pressure BAA-2146 (KpnNDM). The new methodology described right here is quick, strong, scalable, and automated. Our machine for librarypreparation will help within the integration of NGS know-how into all kinds of laboratories, including small analysis laboratories and scientific laboratories.

We have developed an automated high quality management (QC) platform for next-generation sequencing (NGSlibrary characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent supply unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a ready pattern droplet was actuated into place between the bottom electrode and the inlet of the separation capillary to finish the circuit for an electrokinetic injection.

Using a DNA ladder as an inner normal, the CE module with a compact LIF detector was able to detecting dsDNA within the vary of 5-100 pg/μL, appropriate for the quantity of DNA required by the Illumina Genome Analyzer sequencing platform.

This DMF-CE platform consumes tenfold much less pattern quantity than the present Agilent BioAnalyzer QC method, preserving treasured pattern whereas offering obligatory sensitivity and accuracy for optimum sequencing efficiency.

The means of this microfluidic system to validate NGSlibrarypreparation was demonstrated by inspecting the consequences of limited-cycle PCR amplification on the scale distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the scale distribution of the library towards undesirable bigger fragments.

Automated digital microfluidic pattern preparation for next-generation DNA sequencing

Next-generation sequencing (NGS) know-how is a promising device for figuring out and characterizing unknown pathogens, however its usefulness in time-critical biodefense and public well being functions is presently restricted by the dearth of quick, environment friendly, and dependable automated DNA pattern preparation strategies.

To tackle this limitation, we’re growing a digital microfluidic (DMF) platform to operate as a fluid distribution hub, enabling the mixing of a number of subsystem modules into an automatedNGSlibrary pattern preparationsystem.

A novel capillary interface allows extremely repeatable switch of liquid between the DMF machine and the exterior fluidic modules, permitting each continuous-flow and droplet-based pattern manipulations to be carried out in a single built-in system. Here, we spotlight the utility of the DMF hub platform and capillary interface for automating two key operations within the NGS pattern preparation workflow.

Using an in-line contactless conductivity detector at the side of the capillary interface, we display closed-loop automated fraction assortment of goal analytes from a continuous-flow pattern stream into droplets on the DMF machine. Buffer trade and pattern cleanup, probably the most repeated steps in NGSlibrarypreparation, are additionally demonstrated on the DMF platform utilizing a magnetic bead assay and reaching a mean DNA restoration effectivity of 80%±4.8%.

Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing

Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing


Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing

Small ncRNAs (sncRNAs) provide nice hope as biomarkers of illness and response to therapy. This has been highlighted within the context of a number of medical circumstances resembling most cancers, liver illness, heart problems, and central nervous system issues, amongst many others. Here we assessed a number of steps concerned within the improvement of an ncRNA biomarker discovery pipeline, starting from pattern preparation to bioinformatic processing of small RNA sequencing knowledge.


A complete of 45 organic samples have been included within the current research. All libraries have been ready using the Illumina TruSeq Small RNA protocol and sequenced using the HiSeq2500 or MiSeq Illumina sequencers. Small RNA sequencing knowledge was validated using qRT-PCR. At every stage, we evaluated the professionals and cons of totally different strategies which may be appropriate for various experimental designs. Evaluation strategies included high quality of knowledge output in relation to hands-on laboratory time, price, and effectivity of processing.


Our outcomes present that good high quality sequencing libraries could be ready from small quantities of complete RNA and that various degradation ranges within the samples would not have a big impact on the general quantification of sncRNAs by way of NGS. In addition, we describe the strengths and limitations of three commercially obtainable library preparation strategies:

(1) Novex TBE PAGE gel;

(2) Pippin Prep automated gel system; and

(3) AMPure XP beads. We describe our bioinformatics pipeline, present suggestions for sequencing protection, and describe intimately the expression and distribution of all sncRNAs in 4 human tissues: whole-blood, mind, coronary heart and liver.


Ultimately this research gives instruments and end result metrics that can assist researchers and clinicians in selecting an acceptable and efficient high-throughput sequencing quantification technique for numerous research designs, and general producing invaluable info that may contribute to our understanding of small ncRNAs as potential biomarkers and mediators of organic capabilities and illness.

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing

The PacBio RS II supplies for single molecule, real-time DNA know-how to sequence genomes and detect DNA modifications. The start line for high-quality sequence manufacturing is excessive molecular weight genomic DNA. To automate the librarypreparation course of, there should be high-throughput strategies in place to evaluate the genomic DNA, to make sure the dimensions and quantities of the sheared DNA fragments and ultimate library.


The library building automation was completed utilizing the Agilent NGS workstation with Bravo equipment for heating, shaking, cooling, and magnetic bead manipulations for template purification. The high quality management strategies from gDNA enter to ultimate library utilizing the Agilent Bioanalyzer System and Agilent TapeStation System had been evaluated.

 Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing
Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing


Automated protocols of PacBio 10 kb librarypreparation produced libraries with comparable technical efficiency to these generated manually. The TapeStation System proved to be a dependable technique that may very well be utilized in a 96-well plate format to QC the DNA equal to the usual Bioanalyzer System outcomes. The DNA Integrity Number that’s calculated within the TapeStation System software program upon evaluation of genomic DNA is sort of useful to guarantee that the beginning genomic DNA just isn’t degraded. In this respect, the gDNA assay on the TapeStation System is preferable to the DNA 12000 assay on the Bioanalyzer System, which can not run genomic DNA, nor can the Bioanalyzer work instantly from the 96-well plates.

Emulsion PCR (EmPCR) is a generally employed technique for template amplification in a number of NGS-based sequencing platforms. The fundamental precept of emPCR is dilution and compartmentalization of template molecules in water droplets in a water-in-oil emulsion. Ideally, the dilution is to a level the place every droplet accommodates a single template molecule and features as a micro-PCR reactor.

Here, we focus on the fundamental ideas, benefits, and challenges of purposes of emPCR in medical testing. We describe the strategies of preparation and enrichment of template-positive Ion PGM™ Template OT2 200 Ion Sphere™ Particles (ISPs) on the Ion Personal Genome Machine(®) (PGM™) System.

For routine medical testing, following library era, we make use of the automated Ion OneTouch™ System that features the Ion OneTouch™ 2 and the Ion OneTouch™ ES devices for template era and enrichment of template-positive ISPs, respectively.

new analytical technique was developed that integrates a generic pattern preparation into a liquid chromatography-multistage ion lure/time-of-flight mass spectrometry (LC-IT(MS(n))/TOF), allowing for large-scale screening and qualitative confirmation of wide-scope unlawful adulterants in different meals matrices. Samples had been pretreated by a quick single-tube multifunction extraction for correct multistage mass measurement on the hybrid LC-IT/TOF system.

A qualitative validation carried out for over 500 analyte-matrix pairs confirmed the strategy can scale back most of the matrix results and obtain a decrease restrict of confirmation at 0.1 mg/kg for 73% of the goal compounds.

A unique combination of dual-polarity detection, retention time, isotopic profile, and correct MS(n) spectra enables extra comprehensive and exact confirmation, based mostly on the multiparameter matching by automatedlibrary searching against the user-created database. Finally, the applicability of this LC-IT(MS(n))/TOF-based screening process for discriminating coeluting isobars, identifyinnongoal adulterants, and even tentatively elucidating unanticipated species in actual samples is demonstrated.

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