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Category: Molecular and structural bases of living organisms

A “Nano-Robot” Built Entirely from DNA to Explore Cell Processes

Posted on by Léa Surugue

Scientists have designed a “nano-robot” made up of three DNA origami structures. © Gaëtan Bellot/Inserm

Constructing a tiny robot from DNA and using it to study cell processes invisible to the naked eye… You would be forgiven for thinking it is science fiction, but it is in fact the subject of serious research by scientists from Inserm, CNRS and Université de Montpellier at the Structural Biology Center in Montpellier[1]. This highly innovative “nano-robot” should enable closer study of the mechanical forces applied at microscopic levels, which are crucial for many biological and pathological processes. It is described in a new study published in Nature Communications.

Our cells are subject to mechanical forces exerted on a microscopic scale, triggering biological signals essential to many cell processes involved in the normal functioning of our body or in the development of diseases.

For example, the feeling of touch is partly conditional on the application of mechanical forces on specific cell receptors (the discovery of which was this year rewarded by the Nobel Prize in Physiology or Medicine).

In addition to touch, these receptors that are sensitive to mechanical forces (known as mechanoreceptors) enable the regulation of other key biological processes such as blood vessel constriction, pain perception, breathing or even the detection of sound waves in the ear, etc.

The dysfunction of this cellular mechanosensitivity is involved in many diseases – for example, cancer: cancer cells migrate within the body by sounding and constantly adapting to the mechanical properties of their microenvironment. Such adaptation is only possible because specific forces are detected by mechanoreceptors that transmit the information to the cell cytoskeleton.

At present, our knowledge of these molecular mechanisms involved in cell mechanosensitivity is still very limited. Several technologies are already available to apply controlled forces and study these mechanisms, but they have a number of limitations. In particular, they are very costly and do not allow us to study several cell receptors at a time, which makes their use very time-consuming if we want to collect a lot of data.

DNA origami structures

In order to propose an alternative, the research team led by Inserm researcher Gaëtan Bellot at the Structural Biology Center (Inserm/CNRS/Université de Montpellier) decided to use the DNA origami method. This enables the self-assembly of 3D nanostructures in a pre-defined form using the DNA molecule as construction material. Over the last ten years, the technique has allowed major advances in the field of nanotechnology.

This enabled the researchers to design a “nano-robot” composed of three DNA origami structures. Of nanometric size, it is therefore compatible with the size of a human cell. It makes it possible for the first time to apply and control a force with a resolution of 1 piconewton, namely one trillionth of a Newton – with 1 Newton corresponding to the force of a finger clicking on a pen. This is the first time that a human-made, self-assembled DNA-based object can apply force with this accuracy.

 

The team began by coupling the robot with a molecule that recognizes a mechanoreceptor. This made it possible to direct the robot to some of our cells and specifically apply forces to targeted mechanoreceptors localized on the surface of the cells in order to activate them.

Such a tool is very valuable for basic research, as it could be used to better understand the molecular mechanisms involved in cell mechanosensitivity and discover new cell receptors sensitive to mechanical forces. Thanks to the robot, the scientists will also be able to study more precisely at what moment, when applying force, key signaling pathways for many biological and pathological processes are activated at cell level.

“The design of a robot enabling the in vitro and in vivo application of piconewton forces meets a growing demand in the scientific community and represents a major technological advance. However, the biocompatibility of the robot can be considered both an advantage for in vivo applications but may also represent a weakness with sensitivity to enzymes that can degrade DNA. So our next step will be to study how we can modify the surface of the robot so that it is less sensitive to the action of enzymes. We will also try to find other modes of activation of our robot using, for example, a magnetic field,” emphasizes Bellot.

 

[1] Also contributed to this research: the Institute of Functional Genomics (CNRS/Inserm/Université de Montpellier), the Max Mousseron Biomolecules Institute (CNRS/Université de Montpellier/ENSCM), the Paul Pascal Research Center (CNRS/Université de Bordeaux) and the Physiology and Experimental Medicine: Heart-Muscles laboratory (CNRS/Inserm/Université de Montpellier).

A Gene Therapy Studied in Steinert’s Disease

Posted on by Léa Surugue

Steinert’s disease is caused by the abnormal repetitions of a small DNA sequence in the DMPK gene. ©Unsplash

Myotonic dystrophy type 1 (DM1) or Steinert’s disease is a rare and debilitating genetic neuromuscular disease affecting multiple organs and with a fatal outcome. No treatment is available at present. Encouraged by previous research into its molecular causes, researchers from Inserm, CNRS, Sorbonne Université, Lille University Hospital and Université de Lille, in partnership with the Institute of Myology, at the Center for Research in Myology and the Lille Neuroscience & Cognition center, have developed and tested a promising gene therapy that acts directly at the origin of the disease. Initial findings published in Nature Biomedical Engineering show correction of molecular and physiological alterations in mouse skeletal muscle1.

Myotonic dystrophy type 1 (DM1), otherwise known as Steinert’s disease, is a rare, hereditary and genetic neuromuscular condition affecting around 1 in 8,000 people. Debilitating and fatal, it is referred to as a “multisystem” condition because it simultaneously affects the muscles (muscle weakening and atrophy called “dystrophy”; muscle relaxation impairment called “myotonia”) and other organs (cardiorespiratory, digestive and nervous systems, etc.). The expression and course of the disease vary from one patient to the next and no treatment exists as yet.

It is caused by the abnormal repetition of a small DNA sequence (triplet CTG2) in the DMPK (DM1 Protein Kinase) gene located on chromosome 19. In healthy individuals, this sequence is present but repeated between 5 and 37 times. However, in patients with DM1, a mutation occurs whereby the number of triplets increases, producing up to several thousand repetitions.

About the mechanisms enabling gene expression

To obtain the production of a protein, a gene (located in the cell nucleus) is first transcribed into a molecule of RNA. To become a messenger RNA (mRNA), it undergoes maturation, particularly involving splicing. This basically means that the molecule is cut into pieces, some of which are eliminated and others attached. Thanks to this finely regulated process, one gene can lead to the synthesis of different mRNA and therefore of different proteins. After splicing, the mature mRNA will eventually be translated into protein, outside of the cell nucleus.

In Steinert’s disease, the mutated gene is transcribed but the mutant mRNA are retained in the cell nuclei as characteristic aggregates. In the cells of people with DM1, the MBNL1 proteins that normally bind to certain RNA in order to regulate their splicing and maturation are “captured” by the RNA that carry the mutation.

Thus sequestered in the aggregates, it is impossible for them to perform their functions, resulting in the production of proteins that function less well or not at all, some of which have been linked to clinical symptoms.

The team led by CNRS Research Director Denis Furling at the Myology Research Center (Inserm/Sorbonne Université/Institute of Myology) in association with that of Nicolas Sergeant, Inserm Research Director at the Lille Neuroscience & Cognition Centre (Inserm/Université de Lille/Lille University Hospital), focused on a therapeutic strategy to restore the initial activity of MBNL1 in skeletal muscle cells expressing the mutation responsible for Steinert’s disease.

To do this, the scientists engineered modified proteins that present, like the protein MBNL1, binding affinities for the RNA carrying the mutation and as a consequence act as a decoy for these RNA.

When expressing these decoy proteins in vitro in muscle cells from patients with DM1, they observed that they were captured by the mutated RNA instead of the MBNL1 proteins. The latter were then released from the aggregates of mutated RNA and regained their normal function. As a result, the splicing errors initially present in these cells disappeared. Finally, the mutated RNA bound to the decoy proteins proved to be less stable and could be more easily and effectively eliminated by the cell.

Aggregates of mutant DMPK-RNA containing pathological triplet (red) repetitions visualized by FISH-immunofluorescence in the nuclei (blue) of muscle cells (green) isolated from patients with Myotonic dystrophy type1 © Denis Furling and Nicolas Sergeant

The research team then transposed this technique into an animal model in order to verify the validity of this approach in vivo. With the help of viral vectors used in gene therapy, the decoy proteins were expressed in the skeletal muscle of mouse models of Steinert’s disease. In these mice, a single injection was effective, over a long period of time and with few side effects, in correcting the muscle damage associated with the disease, particularly the splicing errors, myopathy and myotonia.

“Our findings highlight the efficacy on Steinert’s disease symptoms of a gene therapy based on the bioengineering of RNA-binding decoy proteins with strong affinity for the pathological repetitions present in the mutated RNA, in order to release the MBNL1 proteins and restore their regulatory functions,” declares Furling. However, the authors point out that additional studies are needed before this therapy can be transposed into a clinical study. “This research paves the way for the development of therapeutic solutions in the context of other diseases in which pathological RNA repetitions cause splicing regulation dysfunction,” concludes Sergeant.

 

1 The striated skeletal muscle is the muscle that is attached to the skeleton by tendons and which, due to its ability to contract, enables the performance of precise movements in a well-defined direction.

2 The coding sequence of a gene consists of the chaining of different combinations of four nucleic acids: adenine, guanine, cytosine, and thymine (replaced by uracil in RNA). These are organized in triplets (or codons) whose correct “reading” by the cell machinery enables the expression of a protein.

Understanding Zebrafish Fin Regeneration Opens up Avenues in Regenerative Medicine

Posted on by Léa Surugue

zebra fish _ poisson zèbre

The zebrafish (Danio rerio) is a tropical species commonly used as a model organism in research laboratories. © Adobe Stock

 

In the animal kingdom, several species share the extraordinary ability to regenerate their limbs or appendages following amputation. One of them is the zebrafish, which is particularly studied in research laboratories due to its ability to regenerate its caudal fin. This phenomenon is made possible by the formation of a blastema, a transient structure composed of undifferentiated cells, which initiates and controls the regeneration of the tissue. Improving our understanding of the cells that make up the blastema and deciphering their interactions opens up new avenues for improving our understanding of the regeneration processes, with the aim of developing clinical applications in the field of regenerative medicine. In a study published in Nature Communications, scientists from Inserm and Université de Montpellier have taken one step closer to this objective, by identifying within the blastema the cell population that coordinates the regeneration process in zebrafish.

The zebrafish, also known as Danio rerio, is a tropical species which has been commonly used as a model organism in research laboratories since the late 1990s. It offers many advantages to scientists, such as the transparency of the embryo and its external development, which is easier to observe than that of mammals. In addition, humans and zebrafish share 70% of the same genes. This genetic conservation with the other vertebrates makes Danio rerio a model of choice for deciphering several major biological processes and their conservation over the course of evolution.

Surprisingly, the zebrafish is also able to regenerate its caudal fin when amputated, thanks to the transient formation of a cell mass known as a “blastema”.

At the larval stage, this structure ensures regeneration of the sectioned appendage in only three days. This is enough to attract the interest of the scientific community, given that understanding the mechanisms associated with this process could pave the way for multiple regenerative medicine applications.

However, only a few cells of the blastema had been described until recently, with the underlying biological mechanisms poorly documented. In their previous work, Inserm Research Director Farida Djouad and her team had highlighted the unique role of macrophages – immune system cells – during the formation of zebrafish blastemas. The team had thus showed that macrophages coordinate the inflammatory processes necessary for the proliferation of the blastema cells and the regeneration of the caudal fin.

 

Identify the cell coordinating the regeneration

In their new study, these researchers went further in exploring the blastema and revealed the major involvement of a new cell population – cells derived from the neural crest[1]. These cells are found in all vertebrates, including in humans, and play a key role in embryonic development.

The scientists deployed several methodological approaches to observe and monitor the fate of the blastema cells. By combining the applications of real-time confocal imaging and single cell RNA-sequencing technology[2] on zebrafish larvae, the Montpellier-based team was able to demonstrate that cells derived from the neural crest coordinate the fin regeneration process, dialoguing with macrophages and other cells in the blastema in order to control and regulate their response. This dialog is conducted via a key factor called NRG1 (Neuregulin 1).

Interactions between the macrophages (in red) and neural crest cells (in green) during regeneration of the zebrafish larva caudal fin. © Farida Djouad

All of these data make it possible to go further in understanding the regeneration processes and their activation in zebrafish. Based on these findings, the next objective will be to understand why mammals, which also possess macrophages and cells derived from the neural crest, fail to regenerate their appendages.

“We are continuing this work on other vertebrate models, including mice, in order to better understand when mammalian embryonic development loses this regeneration capacity, and why, whilst focusing on the role of cells derived from the neural crest,” says Djouad.

“The aim of this research on several animal models capable of regenerating is to identify ‘THE’ coordinating cell, which is common to all regeneration processes. A better understanding of its role, and especially of the factors it secretes, could open up new avenues to promote the regeneration of certain tissues in the treatment of degenerative diseases such as osteoarthritis.”

 

[1] The neural crest of vertebrates is a transient embryonic structure, involved in development, and capable of producing many tissues of the face and skull, particularly the cartilaginous and osteomembranous skeleton, the meninges, the vascular walls of the external and internal carotid system, the dermis… Source: French Academy of Medicine

[2] Single-cell sequencing is based on a set of molecular biology methods to analyze genetic information (DNA, RNA, epigenome…) on a single cell scale.

COVID-19: A New Serological Test to Improve Monitoring of the Pandemic

Posted on by Graziella Cara

Cellules infectées par le SARS-CoV-2

SARS-CoV-2 infected cells. © Sébastien Eymieux and Philippe Roingeard, Inserm – Université de Tours

A new test to detect antibodies to SARS-CoV-2 that is reliable, inexpensive and needs no special equipment? This is the proposal of an international scientific team, of which one of the members is an Inserm researcher at the Institute of Pharmacology and Structural Biology (CNRS/Université Toulouse III – Paul Sabatier). Developed in collaboration with the University of Oxford, this serological test is based on a single reagent that causes red blood cells to agglutinate in the presence of antibodies specific to SARS-CoV-2. Potential initial applications for this test include clinical and epidemiological research. The original article describing the research was published in Nature Communications on March 29, 2021.

Detecting antibodies to SARS-CoV-2 is epidemiologically essential when it comes to monitoring the progression of the epidemic. It is also necessary for scientists studying the links between past contact with the virus and protection against renewed infection. 

Several tests are already available and while effective, they require sophisticated and costly equipment that limits their widespread use, particularly in countries with more limited resources.

That is why Etienne Joly, an Inserm researcher at the Institute of Pharmacology and Structural Biology (CNRS/Université Toulouse III – Paul Sabatier) has devised a new test that is easy to perform and inexpensive. Developed in collaboration with Alain Townsend at the University of Oxford in the UK, the test is based on hemagglutination – a method that has been known for over 50 years and is commonly used to determine blood groups.

The process is based on the agglomeration, visible to the naked eye, of red blood cells in the presence of specific antibodies – in this case directed against SARS-CoV-2. The secret of its simplicity lies in the use of a single reagent consisting of a recombinant protein that associates an antibody recognizing a red-blood-cell surface molecule (glycophorin) with the RBD peptide of the SARS-CoV-2 Spike protein (the domain recognized by the neutralizing antibodies against the virus). When brought into contact with blood, this reagent binds to the red blood cells.

test sérologique Covid-19

The test requires two V-bottomed wells, in which 2 microliters of the blood to be tested are diluted. The left-hand wells – the negative controls – contain the dilution medium, whereas those on the right contain the dilution medium plus the IH4-RBD reagent. After one hour of incubation, the red blood cells have settled, forming a red “button” at the bottom of each well. The plate is then tilted at an 80° angle for 30 seconds and the non-hemagglutinated red blood cells form a teardrop – as can be seen in the left-hand wells. The presence of antibodies in the sample tested on the second line is revealed by the red blood cells agglutinated by the reagent that remain as a “button” at the bottom of the well. © Etienne Joly

 

If, in this same blood sample, antibodies to SARS-CoV-2 are present, they recognize the RBD fragment of the reagent present on the surface of the red blood cells. These antibodies can bind simultaneously to RBD fragments that may be on two different red blood cells, thereby linking the latter together and creating a cluster. Such agglomeration reveals recent or past infection. 

No sophisticated techniques are required for these procedures. The blood can be collected by means of a simple finger-prick, like people with diabetes do when testing their blood sugar levels every day.

Furthermore, the reagent is supplied in lyophilized form that requires no refrigeration and the result can be read with the naked eye. An additional advantage is that this reagent is easy and inexpensive to produce. Its estimated cost of 3 euros per 1000 tests makes it affordable for countries with fewer resources. 

90% sensitivity

The test was evaluated on over 400 serum samples from patients treated in various UK hospitals and presented 90% sensitivity. This means that the test will only detect antibodies in 90 out of every 100 people who possess them. A percentage that Joly is currently working on increasing because it is slightly lower than that of the ELISA tests commonly used in diagnostic laboratories (although it is already better than that of the COVID rapid diagnostic orientation tests available in pharmacies for around ten euros). In terms of specificity, the test achieves 99% – meaning that out of every 100 people who do not possess antibodies, the test will return a false positive for just one of them.

The scientists are now making this test available to interested research laboratories in order to help them elucidate the dynamics of the COVID-19 epidemic. Another advantage is that this test should be easily adaptable to other diseases.

“By modifying the protein of the reagent, it will be possible to tackle the screening of antibodies directed against the variant forms of the virus, or other pathogens such as HIV or the tuberculosis bacterium. We just have to choose the viral or bacterial protein predominantly targeted by antibodies,” concludes Joly.

The “Cocktail Effect” of Endocrine Disruptors Better Understood

Posted on by Graziella Cara

The PXR receptor has a large cavity made up of four pockets (shown in blue, orange, purple and red), which can accommodate several endocrine disruptors at once (their color corresponds to that of the pocket in which they bind). © Vanessa Delfosse

Endocrine disruptors can potentially become more harmful if mixed. Following on from research published in 2015, scientists from Inserm, Université de Montpellier and CNRS at the Structural Biology Center and Montpellier Cancer Research Institute continue to decipher the molecular mechanisms behind this phenomenon known as the “cocktail effect”. While their research provides a better understanding of the complex interactions between endocrine disruptors and the body, it is still in its infancy and must be continued in order to define the real impact of these combinations on human health. Their new study has been published in the journal PNAS.

Scientists continue to elucidate the health effects of environmental pollutants, such as pesticides, residues from medicines, or chemical compounds used in cosmetics and food products. Some of these substances are capable of binding to receptors that are present in or on human cells, in the place of endogenous molecules.

It is at this point that these compounds are referred to as “endocrine disruptors” and may present a risk if they lead to the disruption of certain physiological mechanisms.

The toxicity of several such compounds has already been documented – for example bisphenol A, the exposure to which is linked to an increased risk of certain cancers, metabolic disorders and reduced fertility, or phthalates, which can impair reproductive function.

Researchers are also studying the “cocktail effect”, which is the effect that a mixture of these different substances can have on health. An essential endeavor, given the permanent presence of hundreds of endocrine disruptors in the environment. They rarely act in isolation on human health, but add up and form combinations that can in some cases be harmful.

Two Montpellier-based teams led by Inserm researchers William Bourguet and Patrick Balaguer at the Structural Biology Center (Inserm/CNRS/Université de Montpellier) and the Cancer Research Institute (Inserm/Université de Montpellier) had already discovered that certain endocrine disruptors, which in principle are harmless individually at doses found in the environment, can in some cases be more harmful if mixed.

The scientists had previously shown that two of these compounds, namely 17α-ethinylestradiol (used in certain birth control pills) and TNC (a banned organochlorine pesticide that persists in soils), can bind simultaneously to the same receptor present in the cell nucleus. This receptor, called PXR, controls the expression of different genes involved in regulating various physiological functions.

By binding to this receptor, each of these two endocrine disruptors attracts the other, increasing the amount of product that is bound. An effect that is referred to as “synergistic”, meaning that the function of PXR is altered at much lower doses with this combination of substances than with the individual components, and with a potentially toxic effect.

 

New advances in the understanding of the molecular mechanism

In the new study published in PNAS[1], the researchers went further in understanding this phenomenon by using a method called “crystallography” which makes it possible to observe chemical bonds at the atomic scale, as well as cellular models and in vivo amphibian models. They studied the interactions between the PXR receptor and 13 endocrine disruptors, alone and then in pairs, selected for their affinity with the receptor, their chemical diversity, and their persistence in the environment. The researchers also looked at the impact of these interactions on PXR activity and on the expression of the genes it controls.

They discovered that the PXR receptor actually has four pockets with specific molecular and physico-chemical characteristics. This allows substances with very different structures to interact with and bind to it simultaneously. In addition, PXR exhibits a high level of plasticity, enabling the binding of various unexpected combinations of molecules. By studying the expression of the genes controlled by PXR for each pair of endocrine disruptors that can bind to it, the research teams found that only certain combinations have a strong synergistic effect.

In addition, the researchers also looked at another receptor, RXR, with which PXR combines in order to bind to DNA and regulate gene expression. Using a combination of three endocrine disruptors, they found that the activation of RXR by one of the compounds further reinforced the synergistic effect of the other two PXR-linked disruptors. This mechanism therefore further increases the toxicity of the mixtures.

“This research enabled us to deepen our understanding of the cocktail effect of endocrine disruptors: molecules with a highly variable structure can interact indirectly within the body to obtain mixtures that are toxic to health in both in vitro and animal models, explains William Bourget. And this is just the beginning: while we have discovered a mechanism that explains some of the synergies, these interactions remain complex and others probably exist. These findings do not at this stage make it possible to predict the real impact of these combinations on human health“, he warns.

Although this research focused on PXR, other receptors in cells resemble it and will be the subject of future research by the teams. Ultimately, they hope to elucidate the extent of the phenomenon and above all be able to predict the harmful cocktail effects of several endocrine disruptors. “We are tackling this by combining artificial intelligence with our algorithms. It works for some substances taken alone, but more research is needed into the cocktail effects, which are still very difficult to predict”, concludes Bourguet.

 

[1] The Molecular Physiology and Adaptation laboratory (MNHN/CNRS) and the Hubert-Curien Multidisciplinary Institute (CNRS/Université de Strasbourg) also participated in this new research.

A New Method for Unlocking the Mysteries of Life

Posted on by Graziella Cara

To better characterize molecular interactions, researchers must also look at molecular chirality. © Valérie Gabelica

Understanding the three-dimensional structure of DNA and RNA and how they interact with other molecules is necessary for the advancement of biomedical research and drug development. A team led by Inserm researcher Valérie Gabelica at the Nucleic acids: natural and artificial regulation laboratory (ARNA, Inserm/CNRS/Université de Bordeaux)[1] has developed an innovative method pairing mass spectrometry with circularly polarized light, enabling better characterization of these different molecular interactions. This new technique is described in a study published in the journal Science.

A technique widely used in physics and biology laboratories, and also in forensic science to analyze chemical and biological samples, mass spectrometry measures the masses of each molecule in a sample, thereby providing information on how they interact and associate with each other.

Inserm researcher Valérie Gabelica and her team at the ARNA laboratory (Inserm/CNRS/Université de Bordeaux)1 are studying how short DNA and RNA sequences fold in three dimensions and interact with other molecules. To do this, they use mass spectrometry, which gives them valuable insights into how DNA and RNA associate structurally with proteins or pharmaceutical molecules, for example.

However, to better characterize these molecular interactions, the scientists must also look at molecular chirality. A molecule is said to be “chiral” when its 3D structure cannot be superimposed onto its mirror image. Due to its helical shape, DNA is therefore a chiral molecule.

Determining the chirality of a molecule is important for understanding its biological interactions with DNA and proteins – the molecules of living organisms.

Given that measuring molecular weight provides no information on chirality, the use of another technique, circularly polarized light[2], is necessary to study the 3D structure of molecules.

In a study published in Science, the researcher and her team describe a novel tool for studying chirality and how DNA and molecules assemble: a “2-in-1” method pairing a mass spectrometer and a laser that produces circularly polarized light.

Therapeutic prospects

The use of such a technique could open up new biomedical research avenues, particularly in neurodegenerative diseases, such as Alzheimer’s.

Indeed, several studies have shown that upstream of the protein aggregation process that forms senile plaques in the brain, the proteins form small complexes known as oligomers, which could be even more toxic. Applying the method developed by the researchers would make it possible to improve our understanding of when these oligomers change their 3D structure, in order to explore the role they play in disease exacerbation and plaque formation.

Another practical application is drug development. For example, if the target of a drug is a chiral protein, it is also important to characterize the chirality of the drug in order to find out more about its potential interactions.

“In the 1960s, thalidomide, a drug taken by many pregnant women to reduce nausea, caused severe birth defects in their babies. In fact, one of the images of this molecule in the mirror was anti-nausea, the other was toxic. A more in-depth study of the molecular structure and chirality would have prevented this,” emphasizes Gabelica.

By pushing back hard on the frontiers of mass spectrometry and facilitating the study of molecule chirality, this research is particularly innovative and opens up very broad prospects in the field of biomedical research.

 

[1] The IECB research support unit (CNRS/Université de Bordeaux/Inserm) also participated in this work.

[2]Light is a wave. It is described by signals that oscillate in space and time: electric and magnetic fields. These two fields have a direction, the magnetic field is perpendicular to the electric field. When light is circularly polarized, the electric field changes orientation along the beam and describes a spiral. The magnetic field is always perpendicular to the electric field

Malaria: Vaccine clinical trial for Pregnant Women yields promising results

Posted on by Graziella Cara

©Benoît Gamain. Gestational malaria is associated with low birth weight for the baby and an over-risk of neonatal mortality.

Malaria infection during pregnancy represents a major public health problem in the regions endemic for the disease, substantially increasing the risks to mothers and their unborn children. For newborns, malaria is linked to low birth weight and an excess risk of mortality. To protect this population, a team of researchers from Inserm and Université de Paris led by CNRS Research Director Benoît Gamain is developing a vaccine at the French National Institute of Blood Transfusion (INTS). Called PRIMVAC, the vaccine has undergone a clinical trial to study its safety and collect preliminary data on its ability to induce an immune response. The results of this clinical trial sponsored by Inserm[1] have been published in the prestigious journal Lancet Infectious Diseases.

According to the World Health Organization, malaria is responsible for over 400,000 deaths each year. Despite the progress made in fighting the disease in recent decades, some populations remain particularly vulnerable. One such population is pregnant women.

In the areas of the world where malaria is endemic, people acquire immunity throughout their childhood, meaning that they are generally protected against its most severe outcomes once they reach adulthood. However, pregnant women are an exception because the red blood cells infected with the Plasmodium falciparum parasite responsible for malaria accumulate in the placenta, promoting anemia and gestational hypertension. The disease is also linked to a higher risk of spontaneous abortion, premature birth and intrauterine growth delays which lead to low birth weight and a high rate of neonatal mortality. In Sub-Saharan Africa, 11 million pregnant women were infected with malaria in 2018, with around 900,000 of their babies born underweight.

To tackle this public health problem, a team of researchers from Inserm and Université de Paris led by CNRS Research Director Benoît Gamain has spent the past two decades developing a vaccine for gestational malaria. The goal is to prevent the deaths of up to 10,000 mothers and 200,000 babies each year. “Developing an effective vaccine for young women before their first pregnancy is a priority if we are to reduce malaria-related mortality. An effective strategy could focus on a population similar to that targeted by HPV vaccination, for example, before the women become sexually active”, emphasizes Benoît Gamain.

A safe and effective vaccine

Called PRIMVAC, the vaccine had recently been produced in large quantities in accordance with current regulations. In a clinical trial published in Lancet Infectious Diseases, the researchers provide data on its safety and ability to induce an appropriate immune response, up to 15 months after the initial vaccination.

The vaccine was evaluated in 68 non-pregnant women aged 18 to 35 at the Cochin Pasteur Clinical Investigation Center in Paris, then at the National Center for Research and Training on Malaria (CNRFP) in Ouagadougou, Burkina Faso. The participants were randomly assigned to 4 cohorts, receiving the vaccine at various doses, on 3 occasions over a period of 3 months. These women were then monitored for 15 months in order to identify and treat any side effects and study the immune response induced by the vaccination.

Antibody (green) of a vaccinated volunteer binding to the surface of a human red blood cell infected with the Plasmodium falciparum parasite (blue). Credits: Inserm/Chêne, Arnaud et Semblat, Jean-Philippe

The results of this study show that PRIMVAC is well tolerated. In addition, the researchers have shown that vaccine can produce an immune response, with the production of antibodies in 100% of women vaccinated after only two injections. The antibodies produced are capable of both recognizing the parasitic antigen on the surface of the infected red blood cells and inhibiting their adhesive capacity, which is responsible for their accumulation in the placenta.

“We were able to show that the vaccine is well tolerated, at all the tested doses. The side effects observed were mainly pain at the injection site. We also revealed that the quantity of antibodies generated by the vaccine increases after each vaccination and that they persist for several months. It therefore appears that the vaccine has the capacity to trigger a lasting and potentially protective immune response”, underlines Gamain.

Studying this immune response on the longer term will be the subject of future clinical trials. The researchers want to continue monitoring the 50 Burkinabe volunteers in order to evaluate whether the immune response induced by the vaccination is maintained until their first pregnancy.

 

[1] The trial was coordinated by the Cochin Pasteur Clinical Investigation Center in Paris and the EUCLID/F-CRIN clinical trials platform in Bordeaux in collaboration with the National Center for Research and Training on Malaria (CNRFP) in Ouagadougou, Burkina Faso, and the European Vaccine Initiative (EVI). Funding: Federal Ministry of Education and Research, through the development bank KfW, Germany; Inserm, and National Institute of Blood Transfusion (INTS), France; Irish Aid, Department of Foreign Affairs and Trade, Ireland.

Atopic dermatitis: how allergens get on our nerves

Posted on by Léa Surugue

Mast cells and sensory neurons cluster in “bunches” under the skin. ©Nicolas Gaudenzio

Atopic dermatitis, or eczema, primarily affects infants and children, and manifests itself in hypersensitivity to allergens in the environment. A skin disease characterized by flare-ups, it is often treated with topical anti-inflammatories. A new study led by Inserm researcher Nicolas Gaudenzio, from the Epithelial Differentiation and Rheumatoid Autoimmunity Unit (UDEAR – Inserm / UT3 Paul Sabatier), in collaboration with his colleagues at Stanford University (United States) shows that immune cells and sensory neurons interact in the skin to form units that can detect allergens and trigger inflammation. A discovery that provides an insight into how atopic dermatitis works, and points the way to new therapeutic possibilities. Their findings have now been published in the journal Nature Immunology.

Dry skin, pain, and itching… Atopic dermatitis affects the everyday lives of nearly 20% of children, and up to 5% of adults. The condition can have a significant impact on the quality of life of these patients.

Several studies have shown that genetic factors are involved in the development of this chronic inflammatory skin disease, and suggest that they result in impairment of the skin barrier. This enables the allergens present in the environment, from pollen to dust mites, to penetrate the dermis and stimulate the immune system, which reacts abnormally to this “threat” by triggering eczema.

However, the mechanisms of hypersensitivity to allergens and immune system hyperactivity in patients with atopic dermatitis are not yet fully understood. Led by Inserm researcher Nicolas Gaudenzio, the young “IMMCEPTION” group studies the way in which the immune system interacts with sensory neurons to regulate inflammatory processes in atopic dermatitis.

In particular, the researchers have taken a lead from existing clinical data which show that patients with this disease have numerous neuropeptides in their blood: chemical messengers that carry nerve messages, and whose level is correlated to disease severity. Identification of these neuropeptides in the blood indicates activation of the sensory neurons. These patients also have a number of enzymes in the blood indicating the presence of mast cells. Mast cells are immune cells present in the skin that play an essential role in modulating inflammatory and allergic processes.

Based on these observations, Gaudenzio and his team decided to focus on the interaction between sensory neurons and mast cells, and have now published their findings in the scientific journal Nature Immunology.

The researchers studied animal models of atopic dermatitis. Under the skin of mice showing signs of inflammatory reactions, they observed mast cells and sensory neurons clustering together in “sensory neuroimmune units” not dissimilar in form to a bunch of grapes. “The mast cells and neurons cling together in the dermis. We don’t yet understand the molecular interactions that bind them together, but we have quantified the distances between them, which are tiny,” highlights Gaudenzio.

The researchers then showed that when the mice were exposed to dust mites, these “sensory neuroimmune units” were able to detect the presence of these allergens, triggering allergic inflammation.

In the longer term, this discovery could have practical therapeutic implications.  “Until now, patients could be treated with biological treatments (biological therapy), but these obviously treat the disease further down the line, after flare-ups have occurred. We believe we have put our finger on a trigger mechanism and now want to continue our research to identify new molecules that could block interactions between mast cells and sensory neurons, and thus have a beneficial therapeutic effect for patients,” explains the researcher.

To do so, the group will first need to characterize the molecular interactions within these units in more detail, and analyze the role they play in modulating the immune system.

“One of the questions we are now going to try and answer is what these mast cell-sensory neuron units are for. They must represent a defense mechanism for the body, since they are also found in healthy individuals. But it could be that they don’t work properly in people who have atopic dermatitis—that’s what we’re trying to understand,” concludes Gaudenzio.

This study was funded by the European Research Council (ERC).

Du nouveau dans l’apprentissage automatique via des systèmes biologiques

Posted on by Graziella Cara

 

 

 

The Extraordinary Powers of Bacteria Visualized in Real Time

Posted on by Graziella Cara

Drug resistance in bacterial population. Population of drug resistant bacterial cells producing a fluorescent labelled resistance factor, TetA efflux pump (in red), during treatment with tetracycline antibiotic (in green). Cells producing high levels of TetA efflux pump a capable or effective drug efflux and contain very little tetracycline drug. By contrast, cells containing a lot of tetracycline fail to produce TetA efflux pump.©Christian Lesterlin

The global spread of antibiotic resistance is a major public health issue and a priority for international microbiology research. In his paper to be published in the journal Science, Christian Lesterlin, Inserm researcher at Lyon’s “Molecular Microbiology and Structural Biochemistry” laboratory (CNRS/Université Claude Bernard Lyon 1), and his team were able to film the process of antibiotic resistance acquisition in real time, discovering a key but unexpected player in its maintenance and spread within bacterial populations.

This spread of antibiotic resistance is for the most part due to the capacity of bacteria to exchange genetic material through a process known as bacterial conjugation. The systematic sequencing of pathogenic or environmental strains has identified a wide variety of genetic elements that can be transmitted by conjugation and that carry resistance to most – if not all – classes of antibiotics currently used in the clinical setting. However, the process of transferring genetic material from one bacterium to another in vivo, the time needed to acquire this resistance once the new genetic material is received and the effect of antibiotic molecules on this resistance remained unelucidated.

Real-time visualization

The researchers chose to study the acquisition of Escherichia coli resistance to tetracycline, a commonly used antibiotic, by placing a bacterium that is sensitive to tetracycline in the presence of one that is resistant. Previous studies have shown that such resistance involves the ability of the bacterium to expel the antibiotic before it can exert its destructive effect using “efflux pumps” found on its membrane. These specific efflux pumps are able to eject the antimicrobial molecules from the bacteria, thereby conferring on them a certain level of resistance.

In this experiment, the transmission of the DNA from one specific “efflux pump” – the TetA pump – was observed between a resistant bacterium and a sensitive bacterium using fluorescent marking.  Thanks to live-cell microscopy, the researchers just had to track the progression of the fluorescence to see how the DNA of the “pump” migrated from one bacterium to another and how it was expressed in the recipient bacterium.

The researchers revealed that in just 1 to 2 hours, the single-stranded DNA fragment of the efflux pump was transformed into double-stranded DNA and then translated into functional protein, thereby conferring the tetracycline resistance on the recipient bacterium.

 

The transfer of DNA from the donor bacteria (green) to the recipient bacteria (red) is revealed by the appearance of red localization foci. The rapid expression of the newly acquired genes is revealed by the production of green fluorescence in the recipient bacteria.

How is resistance organized in the presence of an antibiotic?

Tetracycline’s mode of action is well-known to scientists: it kills bacteria by binding to their translational machinery, thereby blocking any possibility of producing proteins. Following this line of reasoning, it would be expected that by adding the antibiotic to the previous culture medium, the TetA efflux pump would not be produced and the bacteria would die. However, the researchers observed that, paradoxically, the bacteria were able to survive and efficiently develop resistance, suggesting the implication of another factor essential to the process of acquiring resistance.

The scientists discovered that this phenomenon can be explained by the existence of another efflux pump that is present in virtually all bacteria: AcrAB-TolC. While this generalist pump is less efficient than TetA, it is still able to expel a small amount of antibiotic from the cell, meaning that the bacteria can maintain minimal protein synthesis activity. Therefore, if the bacterium is lucky enough to have received a resistance gene through conjugation, then the TetA pump is produced and the bacteria becomes durably resistant.

This study opens up new avenues in the search for similar mechanisms in bacteria other than E. coli, and for different antibiotics. “We could even consider a therapy combining an antibiotic and a molecule able to inhibit this generalist pump. While it is still too soon to envisage the therapeutic application of such an inhibitor, numerous studies are currently being performed in this area given the possibility of reducing antibiotic resistance and preventing its spread to the various bacterial species” concludes Lesterlin.

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