Liquid-phase electron microscopy is a technique that can be used to overcome one of the key limitations of electron microscopy – where the electron optics require a high vaccuum, and so the sample would need to be in a stable environment.

Researchers at INM – Leibniz Institute for New Materials, Saarbrücken, Germany, developed this technique in order to study proteins in their hydrated state. The team used a graphene liquid cell for the transmission of proteins during the microscopy, which resulted in an order of magnitude higher radiation tolerance of a sample of protein in comparison to amorphous ice.

Electron microscopy is one of the mainstream methods of analysing and investigating the structure of proteins. Traditionally, during this procedure the protein samples are fixed, then stained with a metal to enhance their contrast, subsequently dried, embedded in plastic, cut in thin sections, and then imaged in the vacuum environment required for electron microscopy.Â

In more recent times, cryo-electron microscopy overcame the intense sample preparation method, by providing the means to study the protein in a native hydrated state – in amorphous ice. In order to resolve the structure, tens of thousands of ‘noisy’ images need to be taken (of identical areas) to resolve the structure of the protein.

The scientists used liquid-phase electron microscopy with unstained protein samples such as nanomaterials or cells in liquid, to image the structure of the protein. Until recently, it was debated whether this method would be better or worse than the method using amorphous ice.

In their recent publication, Dr Sercan Keskin and Dr Niels de Jonge from the INM-Leibniz Institute for New Materials demonstrated how the radiation tolerance was increased by an order of magnitude compared to a sample in ice. The microtubule sample was prepared in a graphene liquid cell, and the researchers mentioned that it was essential to use a low rate when the irradiation electron beam was applied.

The researchers mentioned how this method could potentially overcome limitations observed through other protein structure imaging methods.

Researchers at Purdue University have developed a series of molecules that may provide more reliable relief with fewer side effects for people with any of several autoimmune diseases. The new molecules overcome difficulties with current drugs in targeting, for purposes of inhibiting, the appropriate form of Janus kinase, which has four forms affecting cell signalling and gene expression.

The new inhibitors may provide relief for people suffering from rheumatoid arthritis, psoriasis, myelofibrosis and other autoimmune diseases with a reduction in side effects compared with current therapies. 

“Our new molecules fit within the emerging field of therapeutically useful Janus kinase inhibitors that have attracted a lot of attention and excitement within the medicinal chemistry community and the general field of medicine,” said Mark Cushman, a Distinguished Professor of medicinal chemistry in Purdue’s College of Pharmacy, who leads the research team. “Our compounds contribute a new structural chemotype that is expected to have unique pharmacological properties relative to the other known Janus kinase inhibitors.”

Prof Cushman, a member of the Purdue University Center for Cancer Research, said the new molecules also show potential to allow for more treatment options for people with autoimmune diseases. Abnormalities of the immune system often lead to autoimmune diseases or cancer.

The work aligns with Purdue’s Giant Leaps celebration, celebrating the university’s global advancements in health as part of Purdue’s 150th anniversary. This is one of the four themes of the yearlong celebration’s Ideas Festival, designed to showcase Purdue as an intellectual centre solving real-world issues.

Researchers filed a patent with the Purdue Office of Technology Commercialization and the technology is available for licensing.

The research appears in the Journal of Medicinal Chemistry.

PKCδ

Researchers at The University of Texas MD Anderson Cancer Center have identified a link between an enzyme tied to cancer formation and therapy resistance in patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). They believe two existing therapies may hold promise for clinical studies for this deadly and common lung cancer for which relapse often occurs within one year following treatment.

The study revealed a unique “addiction” between the enzyme protein kinase C delta (PKCδ) found in NSCLC tumors and standard-of-care treatment called EGFR tyrosine kinase inhibitors (TKIs). Based on mouse and human tissue samples, this link allows PKCδ to lessen TKIs’ ability to forestall cancer. 

Approximately 160,000 people in the US are diagnosed with NSCLC each year, and roughly 15,000 have metastatic disease with EGFR mutations.

“Multiple methods of resistance to EGFR TKIs have been identified in EGFR-mutant non-small cell lung cancer, but resistance to treatment remains a major challenge,” said Dr Mien-Chie Hung, chair of Molecular and Cellular Oncology. “Our study identified PKCδ as a common mediator shared by multiple methods of resistance to EGFR TKIs, and it significantly demonstrates inhibition of PKCδ facilitates EGFR TKIs to induce regression of resistant tumors with EGFR mutation.”

EGFR are proteins belonging to a family called receptor tyrosine kinases (RTKs) frequently mutated in NSCLC which cause cellular changes, including enhanced cell growth, tumor formation and metastasis. TKIs interrupt EGFR cell signaling and suppress tumor development. Yet treated tumors eventually develop resistance due to multiple mechanisms known collectively as tumor heterogeneity resistance, which includes additional mutations of EGFR and/or overexpression of other RTKs.

New generations of TKIs targeting specific resistant mechanisms have been developed, which help patients live for several more months, but relapse still occurs and patients eventually are left with no treatment options. To overcome tumor heterogeneity resistance, it is critical to identify a common mediator, which, when blocked, may provide effective treatment, Dr Hung added.

The team observed high levels of PKCδ activation in tumor-bearing mice and patient tissue samples, which revealed PKCδ is a requirement for TKI resistance and is associated with worse progression-free survival following TKI treatment.

The team discovered a combination of two therapies studied in mice, gefitinib and sotrastaurin, appear to be effective as a potential therapy strategy for EGFR-mutant NSCLC TKI resistance. Gefitinib is an FDA-approved TKI for EGFR-mutant NSCLC, while sotrastaurin is approved for use in clinical trials in cancer and other diseases.

“Since gefitinib has received regulatory approval in NSCLC patients and sotrastaurin is available for clinical studies, the combination could be readily tested in clinical trials, especially for patients whose tumor has developed TKI resistance,” said Dr Hung. “It is our hope that new therapies that could result from such studies may be of benefit to patients who have not responded to existing treatments.”

The findings of the study were published in the journal Cancer Cell.

antibiotics

Biomedical engineers at Duke University have shown experimentally that there is more than one flavour of antibiotic resistance and that it could – and should – be taken advantage of to keep first-line antibiotics in our medical arsenal.

In the study, the researchers showed why doctors should be paying more attention to whether a pathogen is resistant or merely resilient against common beta-lactam antibiotics, such as penicillin and its derivatives.

Resistant strains of bacteria can soldier through a dose of beta-lactam antibiotics with little disturbance to their population levels. Resilient strains, however, suffer a population crash before their community can secrete enough beta-lactamase enzymes to degrade the antibiotic to a tolerable level. If clinicians test an infection by dosing a culture and checking only the end results, they miss the difference between resistant and resilient responses.

“Clinicians have not historically distinguished between these two scenarios,” said Dr Lingchong You, the Paul Ruffin Scarborough Associate Professor of Engineering at Duke. “But as beta-lactam-tolerant pathogens become more common, I believe this distinction could become extremely important.”

While an individual bacterium can be resistant to antibiotics, resilience only arises within a community. This happens when bacterial cells produce enough beta-lactamases to degrade the antibiotics, but not enough to save themselves from the initial onslaught. As some cells die and release more and more of the enzyme, however, the population as a whole eventually rids their environment of the antibiotic.

In the study, Prof You and Dr Hannah Meredith, now a postdoctoral researcher at the London School of Hygiene and Tropical Medicine, tracked the populations of several beta-lactam-tolerant strains of bacteria over time when exposed to beta-lactam antibiotics. They then used the responses to quantify the bacteria population’s levels of resistance and/or resilience, creating a method to put values to resistance or resilience for the first time.

In practice, the study offers a framework for researchers to begin designing tests that can swiftly measure an infection for these two separate responses. In You’s opinion, it’s a procedure that will have to become more common in the future.

With a measure of a strain’s resilience in hand, doctors could prescribe a regimen of antibiotics perfectly timed to hit an infection repeatedly during the colony’s weakest points. This approach could allow doctors to continue using first-line antibiotics on pathogens that otherwise would be characterized as resistant and treated with more powerful antibiotics, a practice that will degrade the medicine’s usefulness over time.

“We’re still in a stage where doctors don’t do a detailed diagnosis of what specific infection a patient is suffering from, they just prescribe these antibiotics because they’ll probably work after two weeks. And if they don’t, they’ll just try a different one,” said You. “But I think as these beta-lactam-resistant strains continue to spread around the world and become more common, our diagnoses will have to catch up so we can provide more tailored dosing protocols.”

The study was published in the journal Science Advances.

bacteria

A study has found that any strain of Streptococcus pneumoniae bacteria that is in a mammals tissue first, would be dominant over other competitors of the same species. 

A well-accepted principle in the animal kingdom – from wasps to deer – is that creatures already occupying a habitat nearly always prevail over competitors from the same species that arrive later. Such infighting for the same territory may be deemed “wasteful” by nature.

While the mechanisms that enable “owners” to outcompete “intruders” typically involve a brain that drives such behavior, a new study led by researchers at NYU School of Medicine found this principle to be in play in some bacteria as well – bacteria that declare “ownership” of human host tissue to rival strains and with no brain required.

Results of the study argued that whatever strain of Streptococcus pneumoniae is in place in a mammal’s tissues first is the one more likely to thrive than Strep “latecomers.”

“With Strep infections costing the lives of nearly a million children under five each year globally, we are urgently seeking new ways to defeat bacteria by learning more about how they compete with each other,” said senior study author Dr Jeffrey Weiser, Chair of the Department of Microbiology at NYU Langone Health.

“Nearly all available classes of antibiotics were discovered by studying how microbes kill rivals, including the mechanism used by fungal bread mold to kill bacteria, which yielded penicillin,” said Dr Weiser, also the Jan T. Vilcek Professor of Molecular Pathogenesis.

Taking a step back, past studies have established that bacteria engage certain mechanisms only when an infection matures to the point where the bugs have multiplied beyond a population density threshold, known as a “quorum.”

The new study suggests that among the mechanisms initiated by a Strep quorum is release of two toxins – choline binding protein D (CbpD) and the competence-induced bacteriocins (CibAB). These toxins kill intruding, competing strains. The owners, however, also release other factors that protect them from their own toxins. Their newly arriving relatives, not yet having a quorum, do not yet have their defenses in place.

Still other studies have pointed to the concept that once the intruder bacteria die, they burst and spill their DNA into the extracellular space. This enables the bacteria that killed them to “sample” their DNA, pulling in and using any genes that help the owner to survive, or to defeat antibiotics.

Related to this process is the idea of “competence,” or the ability of a bacterial cell to incorporate foreign DNA, a function that is regulated in Strep by a set of genes called the competence regulon.

Pulling all of these factors together, the new study finds that the mechanism conferring advantage on owner strains is, in fact, the quorum-sensing, competence regulon-induced production and release of bacteriocins like CibAB to kill newly arriving competitors.

Rather than spending energy to have strains fight for dominance, the study authors say, Strep has evolved to fix the fight for the early bird to achieve overall efficiency based on quorum-sensing instead of behavior. This phenomenon could explain the increased instances seen in human infections, where more strains of Strep persist than would be predicted by population models, Prof Weiser said.

The team also used mathematical modeling to support the idea that competence regulon activation has a significant role in competition for dominance, even for strains with different competitive advantages, and that owner dominance manifests quickly.

Within even six hours, for instance, the researchers found that the presence of the owner strain inhibited the long-term presence of, or “colonisation” by, intruders of the same species in the airways of mice, even when the two competing strains had the same genes.

The study was published in Nature Microbiology.

acute myeloid leukaemia

Advances in rapid screening of leukaemia cells for drug susceptibility and resistance are bringing scientists closer to patient-tailored treatment for acute myeloid leukaemia (AML).

Research on the drug responses of leukaemia stem cells may reveal why some attempts to treat are not successful or why initially promising treatment results are not sustained.

AML is a serious disorder of certain blood-forming cells. In this disease, certain early precursor cells in the bone marrow that usually develop into white blood cells don’t mature properly. They remain frozen as primitive cells called blasts, unable to further differentiate and mature. These can accumulate and cause low blood counts that reduce the ability to fight infections, and low platelet counts that cause risk of life-threatening haemorrhage.

Leukaemia stem cells – the progenitors for the immature, cancerous blood cells – propagate AML, and also play a role in cancer returning after treatment. Cancer researchers are interested in how genes are expressed in this cell population, because this data may hold clues to resistance to standard therapies and answers to why some patients relapse.

The study looked at the drug response patterns of stem cells and blast cells taken from individual patients diagnosed with acute myeloid leukaemia. The information was gathered through high throughput screening, a state of the art method for quickly evaluating and testing many samples.

The researchers found that leukaemia stem cells and blast cells diverged in their drug susceptibility patterns, and also that these patterns differed from patient to patient.

For example, blast cells s responded in the test to the drugs most commonly used to treat patients, but none were effective against leukaemia stem cells. The researchers did find 12 drugs from eight classes that seemed to preferentially target leukaemia stem cells, compared to blast cells. Many of them are not often used in patients with this type of cancer.

The multidisciplinary team on the project included stem cell biologists, haematologists, medical oncologists, pathologists, computer scientists, drug developers and others.

The senior researcher was haematologist Dr Pamela Becker, Professor of Medicine at the UW School of Medicine. She is also a scientist at the Fred Hutchinson Cancer Research Center and the UW Medicine Institute for Stem Cells and Regenerative Medicine and sees patients with blood disorders at the Seattle Cancer Care Alliance.

In the laboratory study, the researchers compared the drug sensitivity of blast cell and stem cell populations taken from the same six patients. In doing so, they tested a custom panel of drugs, targeted agents and drug combinations on the cells, and did genetic analyses for 194 mutations. The panel included both FDA approved and investigational drugs.

The unique drug susceptibility patterns observed in leukaemia stem cells and blast cells are leading the scientists to hope that patient-specific approaches could be developed against acute myeloid leukaemia, with the goal of improving the outcomes for people with this form of blood cancer.

virus

No drugs are currently available to treat Ebola, Dengue, or Zika viruses, which infect millions of people every year and result in severe illness, birth defects, and even death. Research from the Gladstone Institutes and UC San Francisco may finally change that. Scientists identified key ways the three viruses hijack the body’s cells, and they found at least one potential drug that can disrupt this process in human cells. What’s more, they discovered how the Zika virus might cause microcephaly in infants, the first step in developing a way to stop the disease.

The researchers employed a technique called protein-protein interaction mapping to probe the three viruses. The method uses human cells in laboratory dishes to create a map of each point of contact between viral and human proteins.

The scientists, whose work was conducted under the umbrella of the Host Pathogen Mapping Initiative launched by the Quantitative Biosciences Institute (QBI) at UCSF, are using these comprehensive maps to target the interactions and try to kill the infection.

By comparing one virus’s map to another, the researchers can find human proteins that are routinely targeted by several different viruses, and that could potentially be involved in other types of human disease as well.

“We’ve employed our systematic protein-protein interaction strategy on Ebola, Dengue, and Zika to get a better sense of how these three very problematic viruses hijack, rewire, and infect human cells,” said the leader of the two studies Dr Nevan Krogan, a senior investigator at the Gladstone Institutes, the director of QBI at UCSF, and a Professor of Cellular and Molecular Pharmacology at UCSF. “To me, what’s most interesting is when we see the same human machinery being hijacked by seemingly very different viruses and different pathogenic proteins.”

By comparing one virus’s map to another, the researchers can find human proteins that are routinely targeted by several different viruses, and that could potentially be involved in other types of human disease as well.

This means targeting those human proteins-which could be considered the weak points of human biology-may be effective in treating many different diseases. For instance, Prof Krogan and his team found that a drug candidate initially explored for cancer treatment can successfully wipe out the infection caused by Dengue and Zika in human cells.

Ratcheting up the Body’s Defenses against Ebola

The group’s first study, conducted in collaboration with Dr Christopher Basler, at Georgia State University, identified 194 virus-human interactions involving six Ebola proteins. The scientists narrowed their focus to one specific point of contact between an Ebola protein called VP30 and a human protein called RBBP6.

The interaction first caught their attention because it was such a strong one, but RBBP6 ended up being a particularly intriguing protein because it has emerged in other virus-protein interaction maps, leading Prof Krogan to believe it plays an important role in the immune system.

It turns out that RBBP6 mimics another Ebola protein called NP. VP30 and NP need to bind to one another in order for the Ebola virus to replicate. However, the human protein RBBP6 interrupts this process by attaching to VP30 instead. So, by blocking the connection between the two Ebola proteins, RBBP6 effectively stops the virus from replicating.

To their surprise, the researchers didn’t discover a way the virus attacks the host (in this case, human cells). Rather, they found a way for the host to fend off the virus.

We often find viral proteins that have evolved to mimic human proteins, but here it’s the opposite

“We often find viral proteins that have evolved to mimic human proteins, but here it’s the opposite,” said Dr Jyoti Batra, one of the first authors of the paper and a postdoctoral scholar at Gladstone who was formerly in Dr Basler’s laboratory at Georgia State University. “It appears our body has a natural way to fight off Ebola infection, and the virus hasn’t gotten around it. Keep in mind, we still don’t have great mechanisms to fight off Ebola, but without this protection the virus would be even deadlier.”

To test this theory, Dr Batra worked with the study’s other first author Dr Judd Hultquist, who conducted the research as a postdoctoral scholar in Prof Krogan’s lab at Gladstone and UCSF and is now an assistant professor at Northwestern University Feinberg School of Medicine. Together, they engineered human cells to either have none of the protein RBBP6 or much higher levels than normal. Then, they infected the cells with the Ebola virus. In cells with no protective protein, infection rates went up fivefold. But the cells with extra protein strongly prevented infection by the virus.

Prof Krogan’s team is now working to identify drugs that can mimic the effects of RBBP6 to fight off Ebola infection.

“This is a key interaction,” said Prof Krogan. “The question is whether we can manipulate it in an effective pharmacological way for it to have therapeutic value.”

Defeating Dengue and Zika

In the second paper, Prof Krogan’s laboratory worked with researchers at Icahn School of Medicine at Mount Sinai and Baylor College of Medicine. They explored the related Dengue and Zika viruses, which are both transmitted by mosquitos and cause similar clinical symptoms.

The scientists hypothesised that if the two viruses interact with human proteins in similar ways, targeting those protein interactions could be the best way to fight the infections. They also mapped the interactions between the Dengue virus and mosquito proteins to compare it to the human-virus protein maps.

“There is something really fascinating about a virus that can do the same thing in two organisms, the human and the mosquito, that have diverged over hundreds of millions of years of evolution,” said Priya Shah, PhD, an assistant professor of chemical engineering and microbiology and molecular genetics at UC Davis who conducted the research while she was a postdoctoral researcher at UCSF. “The virus replicates essentially in the same way in both human and mosquito cells. So, if we can home in on the shared parts of these cells that are exploited by the virus, we could identify a potentially powerful therapeutic target.”

Comparing the three maps, the scientists identified one interaction that occurred in both viruses and both host species: the viral protein NS4A and the host protein SEC61. SEC61 is known to play a critical role in some forms of cancer, and Prof Krogan’s colleague at UCSF, Jack Taunton, PhD, had previously developed compounds targeting these proteins as potential anti-cancer drugs. When the compounds were added to the human and mosquito cells, they effectively wiped out both the Dengue and Zika infections.

We’ve developed an early-stage compound that has very potent antiviral activity against both Zika and Dengue in human and mosquito cells

“We’ve developed an early-stage compound that has very potent antiviral activity against both Zika and Dengue in human and mosquito cells,” said Taunton, a professor in the Department of Cellular and Molecular Pharmacology at UCSF. “Now we need to tweak the molecule to optimise its safety and efficacy before it can be tested in patients.”

“Here’s a great example of repurposing a compound originally developed for one disease and using it to have an effect on another disease,” said Prof Krogan. “It comes back to the bigger picture: by looking across these datasets and across diseases, we can find new connections and innovative solutions.”

Understanding How Zika Causes Microcephaly

Although Dengue and Zika are very similar, only Zika causes the devastating birth defect microcephaly. So, in the final set of experiments, Prof Krogan’s team looked for examples where Zika proteins interacted with human proteins, while Dengue proteins did not.

One interaction that especially stood out was between the Zika protein NS4A and the human protein ANKLE2, which is important for brain development. Mutations in ANKLE2 have previously been linked to hereditary microcephaly.

The scientists found that the Zika protein appears to inhibit the function of ANKLE2, ultimately impairing brain development and leading to microcephaly. The researchers plan to use this knowledge to start developing ways to target ANKLE2 to prevent Zika-related microcephaly.

Looking for Overlaps

The two studies have given the scientists promising leads either to develop new therapies for Ebola, Dengue, and Zika, or repurpose existing ones. By targeting the protein interactions identified in the two studies–especially the human proteins RBBP6 and SEC61-the researchers were able to eradicate all three viruses from human cells, a crucial start in developing potential treatments for the diseases.

The research will continue under the BioFulcrum Viral and Infectious Disease Research Program at Gladstone and Host Pathogen Mapping Initiative at UCSF, which was recently bolstered by a center grant from the National Institutes of Health for $8 million to focus on tuberculosis and Staphylococcus. The two broad-reaching research programs have previously produced protein-protein interaction maps for HIV, tuberculosis, hepatitis C virus, herpesvirus, human papillomavirus, and chlamydia.

“We’re starting to see there’s overlap among the proteins hijacked by different viruses,” said Prof Krogan. “Not only that, but these same proteins are often mutated in diseases with genetic roots, like cancer and autism. The more commonalities we can find between seemingly unrelated diseases, the better we can identify therapies to treat these devastating conditions.”

The papers were published in the journal Cell.

spray

Many people who are diagnosed with cancer will undergo some type of surgery to treat their disease – almost 95 percent of people with early-diagnosed breast cancer will require surgery and it’s often the first line of treatment for people with brain tumors, for example. But despite improvements in surgical techniques over the past decade, the cancer often comes back after the procedure.

A UCLA-led research team has developed a spray gel embedded with immune-boosting drugs that could help. In a peer-reviewed study, the substance was successful half of the time in awakening lab animals’ immune systems to stop the cancer from recurring and inhibit it from spreading to other parts of the body.

The researchers, led by Dr Zhen Gu, a Professor of Bioengineering at the UCLA Samueli School of Engineering and member of the UCLA Jonsson Comprehensive Cancer Center, tested the biodegradable spray gel in mice that had advanced melanoma tumors surgically removed. They found that the gel reduced the growth of the tumor cells that remained after surgery, which helped prevent recurrences of the cancer: After receiving the treatment, 50 percent of the mice survived for at least 60 days without their tumors regrowing.

The spray not only inhibited the recurrence of tumors from the area on the body where it was removed, but it also controlled the development of tumors in other parts of the body, said Prof Gu, who is also a member of the California NanoSystems Institute at UCLA.

The substance will have to go through further testing and approvals before it could be used in humans. But Prof Gu said that the scientists envision the gel being applied to the tumor resection site by surgeons immediately after the tumor is removed during surgery.

“This sprayable gel shows promise against one of the greatest obstacles in curing cancer,” Prof Gu said. “One of the trademarks of cancers is that it spreads. In fact, around 90 percent of people with cancerous tumors end up dying because of tumor recurrence or metastasis. Being able to develop something that helps lower this risk for this to occur and has low toxicity is especially gratifying.”

The researchers loaded nanoparticles with an antibody specifically targeted to block CD47, a protein that cancer cells release as a “don’t-eat-me” signal. By blocking CD47, the antibody enables the immune system to find and ultimately destroy the cancer cells.

The nanoparticles are made of calcium carbonate, a substance that is the main component of egg shells and is often found in rocks. Researchers chose calcium carbonate because it can be gradually dissolved in surgical wound sites, which are slightly acidic, and because it boosts the activity of a type of macrophage that helps rid the body of foreign objects, said Dr Qian Chen, the study’s lead author and a postdoctoral researcher in Prof Gu’s lab.

“We also learned that the gel could activate T-cells in the immune system to get them to work together as another line of attack against lingering cancer cells,” Dr Chen said.

Once the solution is sprayed on the surgical site, it quickly forms a gel embedded with the nanoparticles. The gel helps stop at the surgical site and promotes would healing; the nanoparticles gradually dissolve and release the anti-CD47 antibodies into the body.

The researchers will continue testing the approach in animals to learn the optimal dose, best mix of nanoparticles and ideal treatment frequency, before testing the gel on human patients.

A paper describing the work was published in the journal Nature Nanotechnology.