Using a new system of genetic editing based on bacterial proteins by researchers from MIT cured rare liver disease caused by a single genetic mutation.

The findings described in the edition of Nature Biotechnology, provide the first evidence that the technique of editing of a gene known as CRISPR, can reverse disease symptoms.

CRISPR, which offers an easy way to crop the mutated DNA and replacement with the correct sequence has the potential to treat many genetic diseases, according to the research team.

Recently developed CRISPR system relies on cellular mechanism that bacteria use to protect themselves from viral infection.

Researchers have copied this cell system for the creation of gene-editing complexes, including DNA.

They are cut enzyme called Cas9, bound to the RNA strand, which can be programmed to bind to a specific genomic sequence.

Meanwhile, researchers deliver DNA template strand.

When repairing cell damage resulting from Cas9, scientists introduced the template DNA in the genome.

Scientists predict that this type of revision of the genome one day could help in the treatment of diseases such as hemophilia, Huntington's disease, and the like, caused by a single mutation.

There are other systems developed on the basis of genetic editing of DNA enzymes known as nucleases, but these complexes can be expensive and difficult to assemble.

In contrast, CRISPR is very easy to configure and customize equipment.

Caspase-3 Assay for Apoptosis Detection in Whole, Living Cells 
Catalog no. 9125 (25-50 tests)

Apoptosis is an evolutionarily conserved form of cell suicide mediated by a cascade of proteolytic enzymes called caspases. Pro-apoptotic signals activate the enzymatic cascade resulting in the cleavage of protein substrates, leading to the disassembly of the cell. Caspases have been identified in organisms ranging fromC. eleganstoMembers of the mammalian caspase family of cysteinyl aspartate-specific proteases play distinct roles in apoptosis and inflammation.

Caspases
Active caspase enzymes exhibit catalytic and substrate specificities comprised of short tetra-peptide amino acid sequences that must contain an aspartate in the P1 position. These preferred tetra-peptide sequences have been used to derive peptides that specifically compete for caspase binding. In addition to the distinctive aspartate cleavage site at P1, the catalytic domains of the caspases typically require four amino acids to the left of the cleavage site with P4 as the prominent specificity-determining residue. Addition of a fluoromethyl ketone (FMK) to the tetrapeptide results in an irreversible linkage and permanent inactivation of the cysteine protease enzyme. Furthermore, conjugation of a fluorescent moiety at the amino terminus yields a probe that allows for the detection of caspase activity.

FLICA® 660 Caspase-3 Assay: Detection Mechanism

The far-red FLICA® 660-DEVD-FMK caspase-3 detection probe is comprised of the preferred affinity peptide sequence (DEVD) targeted by activated caspase-3 and caspase-7, a far-red fluorescent 660 dye label, and a fluoromethyl ketone (FMK) reactive moiety. The resulting fluorescent caspase-3 inhibitor probe forms an irreversible, covalent bond with active caspase-3 enzymes, efficiently labeling the target for detection. Due to its cell permeant nature and fluorescence properties, the far-red FLICA caspase-3 detection probe enables whole cell analysis via common fluorescence detection methods.

To use FLICA Caspase-3 Assay, add the caspase-3 detection probe directly to suspension cell or tissue culture media, incubate, and wash. The cell permeant, far-red FLICA caspase-3 detection probe will efficiently diffuse into cells and irreversibly bind to activated caspase-3 enzymes, thereby retaining the red signal inside caspase-3-positive cells. Cells not bearing active caspase-3 return to a non-fluorescent status after the wash step.

The FLICA® 660 caspase-3 detection probe has an optimal excitation at 660 nm and optimal emission range from 680-690 nm. As such, it has demonstrated excellent excitation efficiency with a conventional red HeNe laser with a 633 nm excitation, enabling samples to be analyzed with most flow cytometers and fluorescence microscopes equipped with electronic grey scale image capabilities. Cells labeled with the FLICA® caspase-3 detection probe may be read immediately or preserved for 16 hours using the fixative.

Cat #: 9125 (25-50 tests)

Price: 250 Euro (without VAT)

As the human body fine-tunes its neurological wiring, nerve cells often must fix a faulty connection by amputating an axon -- the "business end" of the neuron that sends electrical impulses to tissues or other neurons. It is a dance with death, however, because the molecular poison the neuron deploys to sever an axon could, if uncontained, kill the entire cell.

Researchers from the University of North Carolina School of Medicine have uncovered some surprising insights about the process of axon amputation, or "pruning," in a study published May 21 in the journal Nature Communications. Axon pruning has mystified scientists curious to know how a neuron can unleash a self -destruct mechanism within its axon, but keep it from spreading to the rest of the cell. The researchers' findings could offer clues about the processes underlying some neurological disorders.

"Aberrant axon pruning is thought to underlie some of the causes for neurodevelopmental disorders, such as schizophrenia and autism," said Mohanish Deshmukh, PhD, professor of cell biology and physiology at UNC and the study's senior author. "This study sheds light on some of the mechanisms by which neurons are able to regulate axon pruning."

Axon pruning is part of normal development and plays a key role in learning and memory. Another important process, apoptosis -- the purposeful death of an entire cell -- is also crucial because it allows the body to cull broken or incorrectly placed neurons. But both processes have been linked with disease when improperly regulated.

The research team placed mouse neurons in special devices called microfluidic chambers that allowed the researchers to independently manipulate the environments surrounding the axon and cell body to induce axon pruning or apoptosis.

They found that although the nerve cell uses the same poison -- a group of molecules known as Caspases -- whether it intends to kill the whole cell or just the axon, it deploys the Caspases in a different way depending on the context.

"People had assumed that the mechanism was the same regardless of whether the context was axon pruning or apoptosis, but we found that it's actually quite distinct," said Deshmukh. "The neuron essentially uses the same components for both cases, but tweaks them in a very elegant way so the neuron knows whether it needs to undergo apoptosis or axon pruning."

In apoptosis, the neuron deploys the deadly Caspases using an activator known as Apaf-1. In the case of axon pruning, Apaf-1 was simply not involved, despite the presence of Caspases. "This is really going to take the field by surprise," said Deshmukh. "There's very little precedent of Caspases being activated without Apaf-1. We just didn't know they could be activated through a different mechanism."

In addition, the team discovered that neurons employ other molecules as safety brakes to keep the "kill" signal contained to the axon alone. "Having this brake keeps that signal from spreading to the rest of the body," said Deshmukh. "Remarkably, just removing one brake makes the neurons more vulnerable."

Deshmukh said the findings offer a glimpse into how nerve cells reconfigure themselves during development and beyond. Enhancing our understanding of these basic processes could help illuminate what has gone wrong in the case of some neurological disorders.