Researchers at Kanazawa University have studied the structure of one of the virulence factors – haemagglutinin (HA) – of avian influenza virus, H5N1. They did this using high-speed atomic force microscopy (HS-AFM) and the findings are essential for developing therapeutic approaches against influenza A viruses in the future.

Understanding the structure and properties of HA when it is initially synthesised by host cells in its precursor form (HA0) is paramount to deciphering HA. So Dr Richard Wong, senior author of the study, and his research team visually analysed the recombinant HA0 protein of H5N1 with the HS-AFM system developed by Kanazawa University.

Both HA0 and HA exist in homotrimeric forms and conversion of HA0 to HA does not significantly modify the homotrimeric structure. Therefore, it is sensible to use HA as a template to generate HA0 HS-AFM simulation images. Acidic endosomal environment is the critical factor for HA to induce fusion between the viral membrane and endosomal membrane in order to release viral materials into host cells.

To elucidate the acidic effect on HA0, it was first exposed to an acidic environment. The trimer of HA0 turned out to be very sensitive to the acidic solution and expanded considerably. When conformational changes of haemagglutinin were measured in real-time using HS-AFM, the team found that its area was larger, and its height shorter. Acidic environment essentially made the molecule flatter and more circular, as compared to its original counterpart. This change in conformation was, however, reversible as the structure reverted back to its original form upon neutralisation.

HS-AFM setup for direct visualisation of HA0 trimer. Schematic diagram of the HS-AFM setup for scanning the HA0 trimer (credit: Kanazawa University).

“Our pilot work establishes HS-AFM as an inimitable tool to directly study viral protein dynamics, which are difficult to capture with low signal-to-noise techniques relying on ensemble averaging, such as cyro-EM and X-ray crystallography,” said lead author of the study Dr Kee Siang Lim. “With high scanning speed and a minimally invasive cantilever, we predict that HS-AFM is feasible to reveal the flow of irreversible conformational changes of HA2 induced by low pH, which is mimicking the true biological events that occur when HA enters a host endosome, in future study.”

This study paved the way for investigating biological events within viruses in real-time. “Our work establishes HS-AFM as an inimitable tool to directly study viral protein dynamics, which are difficult to capture with low signal-to-noise techniques relying on ensemble averaging, such as cyro-EM and X-ray crystallography,” added Dr Richard Wong.

The research can be found on Science Direct.

 

 

 

A study published on 26 April in Science saw that researchers have discovered an acid-activated protein which could prevent tissue damage from stroke, heart attack, cancer and inflammation.

The researchers at John Hopkins University School of Medicine named the newly found protein the proton-activated chloride channel (PAC). The team believes the discovery of this protein could provide a new drug target for potential therapies for stroke and other health issues.

Damage or disease, leading to oxygen deprivation, causes a raised level of acid in tissues. If acidity increases enough the cells become damaged and potentially die. When the build-up of acid reaches a certain level, it opens specialised channels in the cell membrane causing a build-up of ions in the cell, eventually causing it to swell and die. The opening channel has been unidentified until now.

The researchers discovered the channel protein by engineering human cell lines to produce a fluorescent molecule which ceased to glow when the channels in the cell membrane opened in response to acid. In all, 2,725 genes were tested to see which one would open the channels and the gene TMEM206 was found to influence channel activity. When the gene was inactive, it prevented channel activity in response to the acid. This gene matched with a single protein, which the researchers named PAC.

“Knowing the identity of this acid-stimulated protein opens up a broad new avenue of both basic research and drug discovery,” says Zhaozhu Qiu, Ph.D., the study’s principal investigator.

Researchers from King’s College London have shown how skin vaccination can generate protective CD8 T-cells that are recruited to the genital tissues and could be used as a vaccination strategy for STIs.

Before this study, it was thought that vaccines ideally needed to be delivered directly to the body surface where the infection might start, so that the immune system can generate these CD8 T-cells, travel back to the vaccination site and eliminate any future virus that is encountered. However, delivering vaccines directly to the female genital tissue is neither patient-friendly nor efficient.

Now the team from King’s have found that their vaccination strategy marshals a platoon of immune cells, called innate lymphoid cells (ILC1) and monocytes, in the genital tissues to work together and release chemicals (chemokines) to send out a call to the CD8 T-cells generated by the vaccine to troop into the genital tissue.

This research builds on the team’s earlier work to develop skin vaccination techniques using a dissolvable ‘microneedle’ vaccine patch that once placed against the skin dissolves and releases the vaccine without requiring a hypodermic needle injection and generates immune responses.

“This study highlights how specialised groups of ‘innate’ immune cells in distant tissues can be harnessed to attract protective CD8 T-cells, arming the body’s frontline tissues from infection,” said lead author, Professor Linda Klavinskis from King’s. “We now need to confirm these results with other types of vaccines from the one used in the study to see if a common pathway is triggered by skin vaccination.

“If proven, this could have a significant impact in improving the effectiveness of vaccines against sexually transmitted infections.”

The study was published in Nature Communication.

The scientists, funded by the National Institute of Allergy and Infectious Diseases (NIAID), discovered and characterised the structure of a naturally occurring human antibody that recognises and disrupts a portion of the hemagglutinin (HA) protein that the influenza virus uses to enter and infect cells.

The investigators determined that the antibody, FluA-20, binds tightly to an area on the globular head of the HA protein that is only very briefly accessible to antibody attack. 

The team, led by James E. Crowe, Jr, MD, of Vanderbilt University Medical Centre, Nashville, Tennessee, and Ian A Wilson, DPhil., of The Scripps Research Institute, San Diego, California, then isolated FluA-20 antibody from a person who had received many influenza immunisations. Then, in a series of experiments, showed that FluA-20 can ‘reach into’ an otherwise inaccessible part of the three-part HA trimer molecule and cause it to fall apart, thus preventing the spread of virus from cell to cell.

This region of trimeric HA was thought to be stable and inaccessible to antibodies and (unlike the rest of HA’s head) varies little from strain to strain. In theory, antibody-based therapeutics directed at that precise region would be effective against many strains of influenza A virus.

Similarly, vaccines designed to elicit antibodies against this target might provide long-lasting protection against any influenza strain, potentially eliminating the need for annual seasonal influenza vaccination.

In mouse studies, FluA-20 prevented infection or illness when the animals were exposed to four different influenza A viral subtypes that cause disease in humans.

Two viruses used in the experiments, H1N1 and H5N1, are Group 1 influenza subtypes, while the two others, H3N2 and H7N9, are members of Group 2. Current influenza vaccines must contain viral components from both subtypes to elicit matching antibodies. A single vaccine able to generate potent antibodies against members of both groups could provide broad multi-year protection against influenza.

This study, ‘A site of vulnerability on the influenza virus hemagglutinin head domain trimer interface’ by S Bangaru et al. was published in Cell. 

 

 

The researchers’ Y-shaped block catiomer (YBC) binds with certain therapeutic materials to form a package that is 18 nanometers wide – this is less than one-fifth the size of those produced in previous studies, so can pass through much smaller gaps. This allows YBCs to slip through tight barriers in cancers of the brain or pancreas.

A promising field in the fight against cancer is gene therapy, which targets genetic causes of diseases to reduce their effect. The idea is to inject a nucleic acid-based drug into the bloodstream – typically small interfering RNA (siRNA) – which binds to a specific problem-causing gene and deactivates it. However, siRNA is fragile and needs to be protected within a nanoparticle or it breaks down before reaching its target.

“siRNA can switch off specific gene expressions that may cause harm. They are the next generation of biopharmaceuticals that could treat various intractable diseases, including cancer,” explained Kanjiro Miyata, associate professor of the University of Tokyo, who jointly supervised the study. “However, siRNA is easily eliminated from the body by enzymatic degradation or excretion. Clearly a new delivery method was called for.

“We used polymers to fabricate a small and stable nanomachine for the delivery of siRNA drugs to cancer tissues with a tight access barrier,” Miyata continued. “The shape and length of component polymers is precisely adjusted to bind to specific siRNAs, so it is configurable.”

The team’s nanomachine is called a Y-shaped block catiomer because two component molecules of polymeric materials are connected in a Y-shape formation. The YBC has several sites of positive charge which bind to negative charges in siRNA.

The number of positive charges in YBC can be controlled to determine which kind of siRNA it binds with. When YBC and siRNA are bound, they are called a unit polyion complex (uPIC), which are under 20 nanometers in size.

“The most surprising thing about our creation is that the component polymers are so simple, yet uPIC is so stable,” Miyata concluded. “It is early days but I hope to see this research progress from mice to help treat people with hard-to-treat cancers one day.”

 

 

In a preclinical study (published in Cancer Research), biologist Dr Li Zhang and her team at the  University of Texas at Dallas. showed that the expansion of lung tumors in mice slowed when access to heme (the oxygen-binding molecule in hemoglobin) was restricted. The researchers also showed that those same cancers grew faster when more heme was available than normal.

With an eye toward exploiting cancer’s heme dependency, two of Dr Zhang’s graduate students in the Department of Biological Sciences then engineered and extensively characterised new molecules aimed at starving the cancer cells of the molecule that allows them to proliferate so quickly.

The findings suggest a potential new path forward in treating non-small cell lung cancer (NSCLC), which comprises about six out of every seven cases of lung cancer.

However, the team emphasised that its potential treatment approach is designed to work in tandem with chemotherapy or other forms of cancer remedy, not by itself.

“This method wouldn’t kill tumors; it would delay their growth,” explained Dr Zhang. “So it would not be a stand-alone treatment, but it could replace less effective forms of therapy that rely on inhibition of angiogenesis – the creation of new blood vessels.”

These new results emphasise that tumors create energy using molecules other than glucose – a type of sugar known to feed tumors. Where more heme is being made, more oxygen is consumed to make more adenosine triphosphate (ATP) – the energy-carrying molecule that fuels many activities in living cells. Take the heme away, and cancer cell growth slows down.

Sagar Sohoni and Poorva Ghosh, co-first authors of the study, collaborated to investigate how a series of engineered molecules deny heme to cancer cells. These heme-sequestering peptides, or HSPs, are able to ‘hijack’ heme from the spaces between cells, where tumors would be able to access it, while leaving alone the heme in healthy cells.

“Heme sequestering doesn’t adversely affect the normal cells,” Dr Zhang continued. “They synthesise the little heme they need on their own. Also, our peptide won’t go into cells so it shouldn’t provoke many side effects.”

Demonstrating the reduced progress of heme-starved cancer cells makes a compelling case on its own. Dr Zhang’s team also modified NSCLC cells to allow them to obtain heme faster and those cells behaved as expected – aggressive and invasive, consuming oxygen quickly and putting out more ATP.

“Cancer cells’ energy demands are very high compared to the normal cells around them,” Dr Zhang said. “When more heme is available, NSCLC cells grow and reproduce at an alarming rate.”

The next step is to continue refining the design of the HSPs to create a version for use in humans.

Radioactive antibodies are used in nuclear medicine as imaging agents for positron emission tomography (PET) – an imaging technique that improves cancer diagnosis and monitoring chemotherapy. Radioactive drugs can also be designed to kill tumours by delivering a radioactive payload specifically to the cancer cells. This treatment is called targeted radioimmunotherapy.

Conventional methods for radiolabelling proteins are time-consuming and difficult to automate. This new method, developed by Jason P Holland, professor in the Department of Chemistry at the University of Zurich (UZH), uses UV light to synthesise radioactive drugs and diagnostic agents: “By combining photochemistry with radiochemistry, we are now able to make radiolabelled proteins much more quickly and easily,” Holland said.

The team produced a series of novel chemical compounds (chelates) that have two distinctive properties: First, they are able to bind radioactive metal ions like gallium, copper and zirconium. Second, the molecules have a special chemical group that becomes activated by shining UV light on the sample: “The UV light causes the small metal complex to react extremely quickly and efficiently with certain amino acids found in proteins like antibodies,” Holland added.

They were also able to establish a one-pot process to radiolabel trastuzumab, an antibody used to treat patients with breast cancer, with gallium in less than 20 minutes: “The efficient one-pot route has the unique advantage of avoiding the need to isolate and characterise the conjugated intermediate antibody. And the process can be fully automated,” Holland explained further.

Time is one of a huge challenges in radiotracer design for PET imaging since gallium decays rapidly, The research team further developed their method using zirconium to radiolabel trastuzumab. They managed to synthesise the radiolabelled breast cancer antibody in high yield and purity in less than 15 minutes.

With mice bearing human cancer cells that are targeted by trastuzumab, they showed that for PET imaging the zirconium-labelled antibody worked as well as those produced via established methods.

Holland has submitted a patent application for the new procedure and aims to develop the technology along commercial lines. The researchers are also expanding the technology for use against other cancers.

Scientist Michelle Clark, from Rady Children’s Institute for Genomic Medicine, and her colleagues, have built an automated pipeline to analyse EHR data and genome sequencing data from dried blood spots to deliver a potential diagnosis for hospitalised, often critically ill, children with suspected genetic diseases.

Their pipeline required minimal user intervention, increasing usability and shortening time to diagnosis, delivering a provisional finding in a median time of less than 24 hours. Although this pipeline would need to be adapted for use at different hospital systems, such an automated tool could help clinicians diagnose genetic diseases more quickly and accurately, potentially hastening lifesaving changes to patient care.

Genetic diseases are the leading cause of infant mortality in the US, particularly among the around 15 percent of infants admitted to neonatal, pediatric, and cardiovascular intensive care units (ICUs). Rapid disease progression demands an equally fast diagnosis to help inform interventions that lessen suffering and mortality, yet routinely employed genome sequencing takes weeks to return results, which is too slow to guide patient management.

To combat this, Clark and her colleagues analysed the electronic health record (EHR) and genomic sequencing data from both fresh and dried blood samples using a sequencing platform that offered a faster and less labour-intensive approach, as samples could be prepared in batches by an automated robot.

The platform also included a written pipeline of computational scripts that automatically processed the childrens’ EHR data and ranked the likelihood of specific disease-causing variants for each child.

The researchers found that diagnoses matched expert interpretation in 95 children with 97 genetic diseases with 97 percent sensitivity and 99 percent precision. The platform also correctly diagnosed three of seven seriously ill ICU infants with 100 percent sensitivity and precision. 

For further information on the automated pipeline, read the full research article as published in the Science Translational Medicine.

Once the heart is fully formed, the cells that make up heart muscle, known as cardiomyocytes, have limited ability to reproduce themselves. After a heart attack, cardiomyocytes die off and, unable to make new ones, the heart instead forms scar tissue. Over time, this can set people up for heart failure.

MicroRNAs (small molecules that regulate gene function and are abundant in developing hearts), specifically miR-17-92, was identified by Da-Zhi Wang, PhD,  as a regulator of the proliferation of cardiomyocytes in 2013.

In this new work (published in Nature Communications.), Wang (a cardiology researcher at Boston Children’s Hospital and a professor of pediatrics of Harvard Medical School) and his team shows two family members, miR-19a and miR-19b, to be particularly potent and potentially good candidates for treating heart attack.

“The initial purpose is to rescue and protect the heart from long-term damage,” explained Wang. “In the second phase, we believe the microRNAs help with cardiomyocyte proliferation.”

Aside from regulating multiple genetic targets, microRNAs have another advantage as a therapy: they don’t linger in the heart.

“They go in very fast and do not last long, but they have a lasting effect in repairing damaged hearts,” added Jinghai Chen, PhD, a former member of the Wang lab and co-corresponding author on the paper with Wang. “We gave mice only one shot when the heart needed the most help, then we kept checking expression levels of miRNA19a/b post-injection. After one week, expression decreased to a normal level, but the protection lasted for more than one year.”

Even when given systemically, the microRNAs tended to go to the site of heart damage but Wang would like to optimise the specificity of the treatment, since the miRNAs can also affect other tissue and organs.

The next step is to test that treatment in a larger animal before advancing to studies in humans.

Developed by scientists at the New York Genome Centre‘s (NYGC), ECCITE-seq, (Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing) profiles different types of biomolecules from thousands of single cells in parallel. This offers a breadth of information that can be used as readout in CRISPR-based pooled genetics screens. 

“ECCITE-seq is a next-generation tool that enhances our ability to more thoroughly investigate single cells and better understand disease mechanisms,” said NYGC’s Scientific Director and Chief Executive Officer, Tom Maniatis, PhD.

This technique expands from a related tool from 2017 that the Technology Innovation Lab published (named CITE-seq) which enables the detection of proteins together with transcriptomes in single cells.

Then, in 2018, they followed up this work by using the same concept to barcode individual samples, allowing multiplexing of single cell RNA sequencing experiments in a method named Cell Hashing.

“For ECCITE-seq, we adapted our previous work on protein detection and sample multiplexing and combined them with the ability to directly detect single guide RNAs used for CRISPR screens,” added Peter Smibert, PhD, Manager of the NYGC Technology Innovation Lab and corresponding author on the new study. “The separate measurements in ECCITE-seq are modular, so researchers can pick and choose which measurements they need to make for the question they are asking.”

ECCITE-seq is built upon the Single Cell Immune Profiling solution from 10x Genomics, which enables researchers to reconstruct clonotypes of individual immune cells. The team combined protein detection with transcriptomes and clonotypes to characterise malignant populations in a sample from a patient with cutaneous T cell lymphoma.

Combining modalities enabled fine dissection of specific cell subtypes and helped reveal a transcriptomic signature of malignant cells.

“While we have demonstrated the utility of this method in cancer, it is a platform that can be applied to the study of a range of biological systems and diseases,” Dr Smibert continued. “With future developments including the addition of new modalities, we see ECCITE-seq and future methods that expand upon it serving as fundamental tools for better interrogation of individual cells.”

This research was published in Nature Methods.