Author: Megan

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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UFGD develops detergent that eliminates the larvae of the dengue mosquito

Megan Detergent 0 Comment
aedes aegypti mosquitoes

A group of researchers from the Federal University of Grande Dourados, coordinated by Professor Dr. Alexeia Barufatti, are developing a detergent made from the liquid of the cashew nutshell, which in contact with water, eliminates the dengue mosquito larvae. This research is funded by PPSUS through Fundect.

According to Alexeia, the group’s work began in 2015 when the first experiments were carried out with the liquid extracted during the roasting of the cashew nut. The research group is formed by students of scientific initiation, master’s, doctorate, post-doctoral students, in addition to professors from UFGD, UFMS, and UNIFESP.

According to the biologist and postdoctoral fellow at UFGD, Bruno Amaral Crispim, the idea is that the compound is used as a normal detergent in domestic activities and that it would indirectly fulfill the objective of eliminating the larvae of the Aedes Aegypti mosquito.

From the cashew nut oil, we made chemical changes that made this product a detergent, which after tests of the larvicidal effect, proved its effectiveness in combating mosquito larvae. We also carry out tests of environmental effect where we verify that this product when reaching rivers and streams presents little or no toxicity in non-target organisms such as algae, fish, and crustaceans.

cashew nut oil

According to Bruno, the next steps in the project are safety tests for human health and afterward, after the product’s total effectiveness has been proven, make the companies that produce detergents “buy the idea” and start using this compound in their products. products.

ACD RNAscope ISH-IHC

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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

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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

Rotary cell culture system (RCCS)

Megan RCCS 0 Comment
Rotary cell culture system (RCCS)

RCCS is a unique bioreactor technology that produces 3D cultures based on the principle of clinorotation, which is defined as the abolition of gravitational force by slow rotation around one or two axes.

RCCS is a dynamic system that suspends cells without mechanical damage and allows cells to easily aggregate into a 3D spheroid with a sufficient supply of nutrients and oxygen. RCCS can also be used for co-cultivation of several types of cell cultures, cells can be cultured with or without the use of various scaffolds.

It is the first bioreactor system designed to simultaneously integrate the ability to co-cultivate cells and features low shear (and consequently low turbulence) and high nutrient transfer to cells. Together, these properties promote spheroid formation and cell proliferation in three-dimensional spheroids.

Two types of bioreactors: disposable 3D bioreactors and autoclavable 3D bioreactors