RNA

Antitermite Properties of Wood Extracts from Pongamia pinnata (L.) Pierre (Leguminosae) against Subterranean Termites

Megan Detergent, RNA , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 0 Comment
Termites’ mortality was determined in each case of extract and solvent treated Whatman filter paper. Finally, wooden blocks of poplar (19×19×19 mm) were treated with extracts and respective solvents and exposed to termites in the field for 28 days. Minimum mean weight loss was observed in dried P. pinnata (6.38%), followed by fresh P. pinnata in choice tests.
In no-choice tests, dried P. pinnata was comparatively resistant with a weight loss of 12.37%, followed by fresh P. pinnata and P. deltoides. In toxicity bioassay, ethyl acetate-based wood extracts caused the highest mortality (41.66%), followed by petroleum ether, hexane, and water extracts at 10 mg/ml concentration. Similarly, ethyl acetate-based extracts showed maximum repellency (100%) followed by  petroleum ether extracts at 10 mg/ml and ethyl acetate at 5 mg/ml after 60 min of termite exposure. Minimum wood losses were observed in woods treated with ethyl acetate extracts compared to control and other treatments in field experiments.

Isolation of Halomicroarcula pellucida strain GUMF5, an archaeon from the Dead Sea-Israel possessing cellulase

A strain designated GUMF5 was isolated in Goa-India from sediments of Dead Sea-Israel and identified as haloarchaeon Halomicroarcula pellucida based on 16S rRNA gene analysis similarity value of 99.84%. Strain GUMF5 grew on mineral salts medium with 20% NaCl and 0.5% carboxymethyl cellulose-sodium (CMC-Na) as a sole source of carbon and produced haloextremozyme cellulase. The enzyme was concentrated using Sephadex G20, precipitated with ethanol, dialyzed and retentate purified using Sephadex G200, the size exclusion chromatography. A yield of 78.53% cellulase with an activity of 131.13 U/mg and 1.24-fold purity was obtained. More Details
The purified cellulase had optimum activity at 20% NaCl, at 40 ºC, 0.5% CMC-Na, pH 7 and 150 rpm. SDS-PAGE combined with zymographic analysis revealed the molecular weight of cellulase as 240 kDa, 40 kDa and 17.4 kDa. The activity of the enzyme was stimulated by metallic cations in the order of Ca+2 > Mn+2 > Mg+2 > SO4 2- > NH4 + and was inhibited by Ag+ > Fe+2 > Cu+2. Methanol and ethanol enhanced the cellulase activity by 6% and 26%, respectively.
The haloextremozyme cellulase degraded Whatman No. 1 filter paper indicated in scanning electron micrographs, exposure of open pores and fibers without any intra connectivity corresponding to paperase activity and implicating the possible use of enzyme to bio-convert cellulosic waste. Conclusively, Halomicroarcula pellucida GUMF5 (Accession number: MH244431), globally, is the only Halomicroarcula pellucida isolated from the sediments of Dead Sea producing haloextremozyme cellulase, and hence is an important biotechnological resource.

Functional Comparison of Bioactive Cellulose Materials Incorporating Engineered Binding Proteins

Whatman No. 1 chromatography paper is widely used as a substrate for cellulose-based immunoassays. The immobilized proteins are used to capture target biomarkers for detection. However, alternative paper substrates may facilitate mass production of immunoassays as diagnostic tests. Here, we assessed the physical characteristics and protein immobilization capabilities of different commercial papers.
Some substrates fulfilled our design criteria, including adequate flow rate and sufficient protein immobilization for efficient target capture. This study demonstrates that a variety of paper substrates can be bioactivated and used to capture target biomarkers, enabling development of affordable diagnostic tests from a range of starting materials.

Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia

Background: Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection.
This study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia.
Methods: A health facility-based, cross-sectional survey was conducted in selected malaria sentinel sites. Malaria-suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs.
Additionally, five circles of dry blood spot (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at Ethiopian Public Health Institute (EPHI) malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other.
Results: Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, and one mixed infection.
Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI (93-100)) and 83.2% (95% CI (77.6-87.9)), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI 46.9-68.4) and 67% (95% CI 56.2-76.7); and 100% (95% CI 98-100) and 98.9% (95% CI 96-99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65.
Conclusions: Multiplex real-time PCR had an advanced performance in parasite detection and species identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination programme, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

A practical method for storage, preservation and transportation of anuran urine samples using filter paper for hormone analysis

Anurans (frogs and toads) expelled urine when handled and it could provide insights into their physiological status. However, storage, preservation and transportation are often challenging. The study aimed to standardize and validate a field method for short-term storage and preserve of anuran urine samples using Whatman filter papers. To examine the efficacy of storage conditions and type of papers, urinary based enzyme immunoassays were used to measure progesterone and testosterone hormone metabolites.
  • High-Performance Liquid Chromatography was performed and revealed immunoreactive progesterone and testosterone metabolites in the urine samples.
  • Urinary hormone metabolites concentration stored in filter paper at room temperature and control samples stored in -20°C for the same period were similar. 
  • Whatman grade 50 was found to be more suitable for storage of hormones than grade 3 paper for the experiments performed.
  • A cheap and simple storage method for storage of anuran urine in field conditions using filter papers.•Anuran urine could be preserved and transported under ambient conditions without significant changes and loss of hormones.•This method would facilitate the endocrine monitoring of anurans in remote areas where limited logistics are available.

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Direct and Indirect Chemiluminescence: Reactions, Mechanisms and Challenges

Megan RCCS, RNA , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 0 Comment
The phenomenon can take place both in natural and artificial chemical systems and it has been utilized in a variety of applications. In this review, we aim to revisit some of the latest CL applications based on direct and indirect production modes. The characteristics of the chemical reactions and the underpinning CL mechanisms are thoroughly discussed in view of studies from the very recent bibliography.
Different methodologies aiming at higher CL efficiencies are summarized and presented in detail, including CL type and scaffolds used in each study. The CL role in the development of efficient therapeutic platforms is also discussed in relation to the Reactive Oxygen Species (ROS) and singlet oxygen (1O2) produced, as final products.
Moreover, recent research results from our team are included regarding the behavior of commonly used photosensitizers upon chemical activation under  CL conditions. The CL prospects in imaging, biomimetic organic and radical chemistry, and therapeutics are critically presented in respect to the persisting challenges and limitations of the existing strategies to date.

A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase.
In the presence of D-luciferin/ATP assay solution, the new fusion protein displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1-250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein.
Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5-250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

Long-Lasting Luminol Chemiluminescence Emission with 1,10-Phenanthroline-2,9-dicarboxylic Acid Copper(II) Complex on Paper

As most of the known systems are flashtype, long-lasting chemiluminescence (CL) emissions are extremely needed for the application of cold light sources, accurate CL quantitative analysis, and biological mapping. In this work, the flashtype system of luminol was altered to a long lasting CL system just because of the paper substrate. The Cu(II)-based organic complex was loaded on the paper surface, which can trigger luminol-H2O2 to produce a long lasting CL emission for over 30 min.
By using 1,10-phenanthroline-2,9-dicarboxylic acid (PDA) as the ligand, a hexacoordinated Cu(II)-based organic complex was synthesized by the simple freeze-drying method. It is interesting that the complex morphology can be controlled by adding different amounts of water in the synthesizing procedure. The complex with a certain size can be definitely trapped in the pores of the cellulose.
  • Then, slow diffusion, which can be attributed to the long lasting CL emission, was produced. With the high catalytic activity of the complex, reactive oxygen species from H2O2 was generated and was responsible for the high CL intensity.
  • By using the paper substrate, the flash-type luminol system can be easily transferred to the long-duration CL system without any extra reagent.
  • This long-lasting emission system was used for hydrogen sulfide detection by the CL imaging method.
  • This paper-based sensor has great potential for CL imaging in the clinical field in the future.

Insight into the Ozone-Assisted Low-Temperature Combustion of Dimethyl Ether by Means of Stabilized Cool Flames

The low-temperature combustion kinetics of dimethyl ether (DME) were studied by means of stabilized cool flames in a heated stagnation plate burner configuration using ozone-seeded premixed flows of DME/O2. Direct imaging of CH2O* chemiluminescence and laser-induced fluorescence of CH2O were used to determine the flame front positions in a wide range of lean and ultra-lean equivalence ratios and ozone concentrations for two strain rates.
The temperature and species mole fraction profiles along the flame were measured by coupling thermocouples, gas chromatography, micro-chromatography, and quadrupole mass spectrometry analysis. A new kinetic model was built on the basis of the Aramco 1.3 model, coupled with a validated submechanism of O3 chemistry, and was updated to improve the agreement with the obtained experimental results and experimental data available in the literature.
The main results show the efficiency of the tested model to predict the flame front position and temperature in every tested condition, as well as the importance of reactions typical of atmospheric chemistry in the prediction of cool flame occurrence. The agreement on the fuel and major products is overall good, except for methanol, highlighting some missing kinetic pathways for the DME/O2/O3 system, possibly linked to the direct addition of atomic oxygen on the fuel radical, modifying the product distribution after the cool flame.

Advanced image analysis-based evaluation of protein antibody microarray chemiluminescence signal improves glioma type identification by blood serum proteins concentrations

Background and objective: Gliomas are the most common brain tumors usually classified as benign low-grade or aggressive high-grade glioma. One of the promising possibilities of glioma diagnostics and tumor type identification could be based on concentration measurements of glioma secreted proteins in the blood.
However, several published approaches of quantitative proteomic analysis emphasize limits of one single protein to be used as biomarker of these types of tumors. Simultaneous multi-protein concentrations analysis giving antibody array-based methods suffer from poor measurement accuracy due to technical limitations of imaging systems.
Methods: We applied Principal Component Analysis (PCA) for series of repeated antibody array chemiluminescence images to extract the component representing relative values of protein concentrations, free from zero-mean noise and uneven background illumination – main factors corrupting evaluation result.
Results: The proposed method increased accuracy of protein concentration estimates at least 2-fold. Decision tree classifier applied to the relative concentration values of three proteins TIMP-1, PAI-1 and NCAM-1 estimated by proposed image analysis method effectively distinguished between low-grade glioma, high-grade glioma and healthy control subjects showing validation accuracy of 74.9% with the highest positive predictive value of 81.2% for high-grade glioma and 57.1% for low-grade glioma cases.

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The kidney releases a non-polymerizing form of Uromodulin in the urine and circulation that retains the external hydrophobic patch domain

Megan RNA , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 0 Comment
Uromodulin (Tamm-Horsfall protein, THP) is a glycoprotein uniquely produced in the kidney. It is released by cells of the thick ascending limbs (TAL) apically in the urine, and basolaterally in the renal interstitium and systemic circulation. Processing of mature urinary THP, which polymerizes into supra-molecular filaments, requires cleavage of an external hydrophobic patch (EHP) at the C-terminus. However, THP in the circulation is not polymerized, and it remains unclear if non-aggregated forms of THP exist natively in the urine.
We propose that an alternative processing path, which retains the EHP domain, can lead to a non-polymerizing form of THP. We generated an antibody that specifically recognizes THP with retained EHP (THP+EHP) and established its presence in the urine in a non-polymerized native state. Proteomic characterization of urinary THP+EHP revealed its C-terminus ending at F617. In the human kidney, THP+EHP was detected in TAL cells, and less strongly in the renal parenchyma.
Using immunoprecipitation followed by proteomic sequencing and immunoblotting, we then demonstrated that serum THP has also retained EHP. In a small cohort of patients at risk for acute kidney injury (AKI), admission urinary THP+EHP was significantly lower in patients who subsequently developed AKI during hospitalization. Our findings uncover novel insights into uromodulin biology by establishing the presence of an alternative path for cellular processing, which could explain the release of non-polymerizing THP in circulation joplink Immobilized Papain. Larger studies are needed to establish the utility of urinary THP+EHP as a sensitive biomarker of kidney health and susceptibility to injury.

A crystal-processing machine using a deep-ultraviolet laser: application to long-wavelength native SAD experiments

While native SAD phasing is a promising method for next-generation macromolecular crystallography, it requires the collection of high-quality diffraction data using long-wavelength X-rays. The crystal itself and the noncrystalline medium around the crystal can cause background noise during long-wavelength X-ray data collection, hampering native SAD phasing. Optimizing the crystal size and shape or removing noncrystalline sample portions have thus been considered to be effective means of improving the data quality. A crystal-processing machine that uses a deep-UV laser has been developed.
The machine utilizes the pulsed UV laser soft ablation (PULSA) technique, which generates less heat than methods using infrared or visible lasers. Since protein crystals are sensitive to heat damage, PULSA is an appropriate method to process them. Integration of a high-speed Galvano scanner and a high-precision goniometer enables protein crystals to be shaped precisely and efficiently. Application of this crystal-processing machine to a long-wavelength X-ray diffraction experiment significantly improved the diffraction data quality and thereby increased the success rate in experimental phasing using anomalous diffraction from atoms.

Development of high-resolution multidimensional native protein microfluidic chip electrophoresis fingerprinting and its application in the quick analysis of unknown microorganisms

The unascertained, constant mutation and emergence of new types of microorganisms present significant challenges to their detection. Differing from the focus on the limited local 16S rRNA gene or protein markers, characteristic whole fingerprint technologies at the omic level are particularly suitable for unknown analytes since accurate knowledge about the constituents is not necessarily required.
Herein, through a combination of several innovative strategies, including pure water isotachophoresis integrated (2 + 1)D electrophoresis, inversion-funnel peak stacking channel geometry and COMSOL computer-aided fluid simulation, high-resolution whole protein 2D native microfluidic chip electrophoresis was achieved within less than 1 min. The highest ever reported peak capacity for native 2D chip electrophoresis was obtained.
Furthermore, taking Escherichia coli, Staphylococcus aureus, and Bacillus subtilis as model analytes without protein biomarker information, the feasibility of the identification and semiqualification of unknown microbes in pure or mixed samples was explored with the utilisation of original algorithms, including SIFT feature abstraction and a global information entropy combined support vector machine.
As such, the multidisciplinary cooperation in the present study demonstrates monstrated promising prospects for microfluidic chip electropherogram fingerprint-based quick microorganism assays, biointeraction studies, and drug screenings, even if the analytes are not fully ascertained.

Drug targeting opportunities en route to Ras nanoclusters

Disruption of the native membrane organization of Ras by the farnesyltransferase inhibitor tipifarnib in the late 1990s constituted the first indirect approach to drug target Ras. Since then, our understanding of how dynamically Ras shuttles between subcellular locations has changed significantly. Ras proteins have to arrive at the plasma membrane for efficient MAPK-signal propagation. On the plasma membrane Ras proteins are organized into isoform specific proteo-lipid assemblies called nanocluster.
Recent evidence suggests that Ras nanocluster have a specific lipid composition, which supports the recruitment of effectors such as Raf. Conversely, effectors possess lipid-recognition motifs, which appear to serve as co-incidence detectors for the lipid domain of a given Ras isoform. Evidence suggests that dimeric Raf proteins then co-assemble dimeric Ras in an immobile complex, thus forming the minimal unit of an active nanocluster.
Here we review established and novel trafficking chaperones and trafficking factors of Ras, along with the set of lipid and protein modulators of Ras nanoclustering. We highlight drug targeting approaches and opportunities against these determinants of functional Ras membrane organization. Finally, we reflect on implications for Ras signaling in polarized cells, such as epithelia, which are a common origin of tumorigenesis.

The native state conformational heterogeneity in the energy landscape of protein folding

The native structure of proteins is central to various functions performed by cells. A vital part of the structure-function paradigm of proteins is their inherent flexibility and dynamics. The dynamic interconversion between the conformational substates in the heterogeneous native state basin of the energy landscape enables a single protein molecule to perform multiple functions. The dynamics among the substates are assisted by the motion of different structural elements of a protein out of which side-chains of amino acids hold a significant position due to their involvement in various functions such as molecular recognition and dynamic allostery.
This review briefly describes the origin of conformational heterogeneity in the native state ensemble and the motions of different structural modules that assist the equilibrium dynamics of the conformational substates. The review then centers the discussion on conformational heterogeneity due to side-chain movements in proteins, the experimental methods to detect and characterize them, and their role in performing multiple functions.

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ACD RNAscope ISH-IHC

Megan RNA 0 Comment
ACD RNAscope ISH-IHC

2 wishes to satisfy
Transcriptomics & Proteomics

  • It can simultaneously detect the expression of RNA and Protein in a single cell in the same sample, and retain tissue morphological information.
  • Overcoming the problems of low sensitivity and low specificity of traditional ISH, RNAscope has the advantages of high sensitivity, high specificity, time-saving and easy operation, allowing RNAscope ISH-IHC to get better results
  • Can be applied to: Immuno-oncology, Neurobiology, Cell, Gene Therapy & IHC Validation

Three precautions before implementing Dual ISH-IHC/IF

  • Confirm that RNAscope ISH can get good results
  • Confirm that antibodies can get good results in IHC/IF
  • Since the pre-processing of RNAscope will use Protease, it is recommended to use Protease for pre-processing before testing IHC/IF experiments to confirm antibodies and proteins The binding position will not be affected, the IHC/IF signal has not weakened, and the background value has not increased (as shown below).

RNAscope Duplex + IHC

RNAscope VS Duplex combined with IHC to detect the expression of immune cells chemokines and cytokines in human lung tumors

(A) Detect the RNA of IL-12 and CXCL9 and the macrophage Marker CD68 protein
(B) Detect the RNA of TGFB and FOXP3 and the T cell Marker CD4 protein.

RNAscope HIplex + IHC

Dual ISH-IHC/IF can detect different neuronal subtypes and detect the expression of circRNA splice variants in specific cells.

RNAscope HiPlex can detect up to 12 target RNAs; the figure simultaneously detects the performance of Drd1 + and Drd2 + striatal neuron subtypes, neuron Marker NeuN protein (white) and circRNA splice variants.

SiRNA & shRNA Gene Silencers

Megan RNA 0 Comment
SiRNA & shRNA Gene Silencers

RNAi (RNA interference): The combination of small fragments of nucleotide molecules with complementary mRNAs leads to degradation of the mRNA and gene knockdown. The Santa Cruz RNAi system includes siRNA, shRNA Plasmid and shRNA Lentiviral particles. The product line covers more than 99% mouse and human protein-coding genes.

● siRNA gene silencers
consist of three to five pairs of 19-25nt (nucleotide), and the concentration of 10µM can provide 50-100 transfections

● shRNA plasmid DNA
uses Hi promote to make shRNA stable and continuous performance. With puromycin resistant, antibiotics can be used for screening

● shRNA Lentiviral particles
use viral infection to enter cells

Common Q&A

Q1: What are the main differences between shRNA Lentiviral Particles and shRNA Plasmids?
A1: shRNA Lentiviral Particles use viral infection to enter cells. It is recommended to use primary cells that are not easy to transfect.

Q2: Are the siRNA and shRNA sequences identical?
A2: Yes

Q3: How to confirm the effect of transfection?
A3: The transfection or transduction efficiency can be confirmed by the green fluorescence of copGFP plasmid (sc-108083) and copGFP Lentiviral Particles (sc-108084)

Q4: What is the difference between the shRNA plasmid item number (h) and (h2)?
A4: (h) and (h2) are used to silence the same gene, but the sequence is different.

Q5: Can you provide the sequence of siRNA or shRNA?
A5: Yes, just provide the batch number after ordering!

Q6: Can lentiviral vector plasmid be amplified by itself?
A6: No

Q7:shRNA contains 3-5 plastids? Can they be purchased separately?
A7: The original factory currently only provides siRNA sequences for purchase separately