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Viruses are parasites. The only way they can grow is by hijacking their hosts. When they infect a human host, viruses use human proteins to multiply and modify the human cells to sustain the infection. At the same time, the human host activates defense mechanisms to fight the infection.

Most current drugs against viral infections target the virus itself. But scientists are interested in developing therapies that aim for host proteins instead, or the genes that produce them, in part because such therapies are believed less likely to elicit drug resistance. A detailed understanding of virus-host interactions is crucial to the success of this strategy.

A team of Gladstone Institutes scientists led by Senior Investigator Nevan Krogan, PhD, has been cataloging host proteins that physically bind to virus proteins. These physical interactions identify human proteins that the virus can use to infect cells and propagate. However, they don't reveal how host proteins work together to facilitate infection.

To address this gap, Krogan and staff scientist David Gordon, PhD, with colleagues at UC San Francisco (UCSF), University College Dublin, and the Mount Sinai School of Medicine, have developed a new way to understand how host cells control HIV infection in human cells.

Their approach entails disrupting host genes rather than proteins. It is based on the idea, pioneered by Krogan, that you obtain richer information about the functions of genes–and the proteins they encode–when you disable the genes in pairs, instead of one by one. In a paper published in Molecular Cell, the team describes a map of the genes controlling HIV infection in human cells, which they built by assessing more than 63,000 combinations of human genes associated with HIV infection.

HIV is a major public health concern, with an estimated 36.7 million people living with chronic infection, and over 20.9 million people receiving continuous treatment. Studying the impact of gene disruptions in pairs rather than one by one yields important information on how genes work together to mediate virus infection, highlighting processes we can target with drugs to inhibit infection."

Nevan Krogan, Ph.D., senior investigator, professor of Cellular and Molecular Pharmacology at UCSF, and the director of the UCSF Quantitative Biosciences Institute

The map, which the team refers to as a viral epistasis map (vE-MAP), is an essential advance for HIV research in several other ways. For one thing, it uncovers a previously unsuspected set of genes required for the growth of the virus in human cells. For another, the vE-MAP can be used to analyze how different HIV mutants affect host cells or to test drugs that disrupt HIV-host interactions.

Strength in numbers

The vE-MAP is an adaptation of the E-MAP, which Krogan and his colleagues pioneered and refined over the past 15 years to identify genes that control how cells grow. At the core of this approach is the Krogan lab's ability to disrupt a large number of genes, test them in pairs, and analyze the results via sophisticated computational methods.

"The principle behind E-MAPs is that when you disrupt two genes at once and examine the impact on a cell, you sometimes see effects that are significantly larger or smaller than you would have predicted from the effect of disrupting either gene alone," said Krogan.

These unexpected effects suggest that the functions of the two genes are related. Moreover, by carrying out these pairwise disruptions across hundreds of genes, scientists can find groups of genes with similar patterns of interactions, a sign that they are likely to take part in the same molecular process.

"And so, instead of finding important genes one at a time, you can all at once identify multiple, distinct networks of genes affecting the process you are studying," said Gordon.

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The E-MAP approach has mostly been used to study cell growth. Gordon, in collaboration with a student from University College Dublin, Ariane Watson, had to modify it to study virus infection. The most tricky part was to implement a sophisticated data acquisition and scoring system, which allowed them to measure HIV infection accurately across hundreds of thousands of samples, and compare the effect of pairwise and single-gene disruptions.

It would be an overwhelming effort to test all combinations of the over 20,000 protein-coding genes in the human genome. Instead, the scientists focused on genes already suspected to influence HIV biology. In particular, they used the genes encoding a large number of human proteins that the Krogan lab had previously found to bind to HIV proteins. In all, they included over 350 genes in their analysis and tested over 63,000 pairwise disruptions.

New players at the HIV-host interface

Although HIV is one of the best-studied human viruses and is now well-controlled by antiretroviral therapy, there is no cure for HIV/AIDS. Moreover, antiretroviral therapy is costly, which can make it impractical in resource-poor countries. The search for new means of halting or eradicating the virus is, therefore, still a priority.

Among the genes that stood out in the vE-MAP were several members of the CNOT family, whose role in HIV biology had never before been established. The authors demonstrated that the CNOT complex promotes HIV infection by suppressing innate immunity in CD4+ T cells, the type of immune cells that HIV preferentially targets in humans. Innate immunity is a defense mechanism by which host cells can fight infection.

"The impact of CNOT on innate immunity is a key, yet previously unrecognized, host pathway critical to HIV infection. It will serve as a potential novel therapeutic target in future studies," said Krogan.

For instance, scientists can now study if targeting the CNOT complex with drugs could be a way to help HIV patients fight the infection more effectively.

Furthermore, the vE-MAP uncovered genes that had little impact when disrupted individually, but a great effect when tested together.

"These genes would be overlooked in classic, single-gene disruption experiments," said Gordon. "They confirm the potential of the vE-MAP to uncover new mechanisms by which HIV interacts with human cells."

Combining drugs that target two of these genes at the same time might thus be a promising therapeutic strategy, especially for a virus such as HIV/AIDS, which has evolved multiple ways of tapping its hosts' resources.

The vE-MAP was also able to pick up genes that specifically interact with a known HIV mutant. This observation bodes well for the ability of the vE-MAP to identify distinct host factors affecting the various forms of HIV, or the virus mutants that arise in response to currently available drugs.

Additional testing with a drug known to interfere with HIV-associated human proteins gives the authors confidence that their vE-MAP approach could, in the future, be used to screen for novel anti-HIV drugs and to understand their mode of action.

"This vE-MAP provides an unprecedented view of how HIV hijacks and rewires the cellular machinery in human cells during infection," said Krogan. "It will generate many new ideas and avenues to identify and test novel therapies."

And the benefits may not be limited to HIV research.

"Our work is proof-of-principle that the vE-MAP approach is a powerful way to map out the interface between HIV and human cells, and to uncover new therapeutic avenues," said Gordon. "We now look forward to testing it on other pathogens."

Source:

Gladstone Institutes

Researchers at the University of California, Riverside, have completed a cross-sectional human study that compares biomarkers and metal concentrations in the urine of e-cigarette users, nonsmokers, and cigarette smokers.

They found that the biomarkers, which reflect exposure, effect, and potential harm, are both elevated in e-cigarette users compared to the other groups and linked to metal exposure and oxidative DNA damage.

Our study found e-cigarette users are exposed to increased concentrations of potentially harmful levels of metals — especially zinc — that are correlated to elevated oxidative DNA damage.”

Prue Talbot, professor of cell biology, University of California, Riverside

Zinc, a dietary nutrient, plays key roles in growth, immune function, and wound healing. Too little of this essential trace element can cause death; too much of it can cause disease. Its deficiency, as well as its excess, cause cellular oxidative stress, which, if unchecked, can lead to diseases such as atherosclerosis, coronary heart disease, pulmonary fibrosis, acute lymphoblastic leukemia, and lung cancer.

Electronic cigarettes consist of a battery, atomizing unit, and refill fluid. Metals in e-cigarette aerosols come mainly from the metal components in the atomizer– nichrome wire, tin solder joints, brass clamps, insulating sheaths, and wicks — as well as the e-fluids that the atomizers heat.

The study, which appears in BMJ Open Respiratory Research, marks the first time researchers have examined and quantified urinary biomarkers of effect and potential harm in relation to metals in e-cigarette users.

A biomarker is a quantifiable characteristic of a biological process. Biomarkers allow researchers and physicians to measure a biological or chemical substance that is indicative of a person’s physiological state. Previous e-cigarette studies with humans have examined biomarkers of exposure — for example, nicotine or nicotine metabolites — but none have studied biomarkers of potential harm or shown how this harm correlates with metal exposure.

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The biomarkers studied by the UC Riverside researchers were 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage; 8-isoprostane, an indicator of the oxidative degradation of lipids; and metallothionein, a metal response protein. All three biomarkers were significantly elevated in e-cigarette users compared to the concentrations in cigarette smokers.

“Our findings reaffirm that e-cigarette use is not harm free,” said Shane Sakamaki-Ching, a graduate student in the Cell, Molecular and Developmental Biology Graduate Program and the research paper’s first author. “Indeed, prolonged use may lead to disease progression.”

The researchers advise physicians to exercise caution when recommending e-cigarettes to their patients. Electronic cigarette aerosols contain potentially harmful chemicals, cytotoxic flavor chemicals, metals, ultrafine particles, and reaction products. E-cigarette use has been linked to adverse health effects such as respiratory diseases, increased risk for cardiovascular disease, and impaired wound healing following surgery.

“Pregnant women, especially, should not be encouraged to use e-cigarettes,” Talbot said. “Excess of zinc in their bodies can lead to nausea and diarrhea. Given the recent deaths and pulmonary illnesses related to e-cigarette usage, everyone should be made aware of the potential health risks linked to e-cigarette usage.”

Source:

University of California – Riverside

Journal reference:

Sakamaki-Ching, S., et al. (2020) Correlation between biomarkers of exposure, effect and potential harm in the urine of electronic cigarette users. BMJ Open Respiratory Research. doi.org/10.1136/bmjresp-2019-000452.

After 50 years of research and the testing of over 1,000 drugs, there is new hope for preserving brain cells for a time after stroke. Treating acute ischemic stroke patients with an experimental neuroprotective drug, combined with a surgical procedure to remove the clot improves outcomes as shown by clinical trial results published today in The Lancet.

The multi-center, double-blinded, randomized trial, led by a team at the Cumming School of Medicine's (CSM) Hotchkiss Brain Institute and Alberta Health Services, investigates the use of the neuroprotective drug nerinetide, developed by NoNO Inc, in two scenarios in the same trial. In one scenario, nerinetide is given to patients in addition to the clot-busting drug alteplase. In the second scenario, patients who were not suitable for alteplase received only nerinetide. Both groups of patients had concurrent endovascular treatment (EVT) to remove the clot.

"Compared to placebo, almost 20 per cent more patients who received nerinetide along with endovascular treatment, but did not receive alteplase, recovered from a devastating stroke – a difference between paralysis and walking out of the hospital," says Dr. Michael Hill, MD, a neurologist at Foothills Medical Centre (FMC) and professor in the departments of Clinical Neurosciences and Radiology at the CSM. "In the patients who received both drugs, the alteplase negated the benefits of the nerinetide."

Hill says the study provides evidence of a biological pathway that protects brain cells from dying when they are deprived of blood flow. Nerinetide targets the final stage of the brain cell's life by stopping the production of nitric oxide within the cell.

"We really believe this is a new scientific observation," says Hill. "There is evidence nerinetide promotes brain cell survival, offering neuroprotection until we can extract the clot. It opens the door to a new way of treating stroke."

Images of patients' brains from the study show the expected size of the damage from the stroke is sizeably reduced when nerinetide is administered and EVT is performed among patients not concurrently receiving alteplase.

After so many studies investigating neuroprotective drugs failed, we are extremely excited by these results. While nerinetide is not approved for use yet, it shows the potential of a new tool to promote recovery from stroke."

Dr. Mayank Goyal, MD, PhD, neuroradiologist at the FMC, and clinical professor in the Department of Radiology at the CSM

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Worldwide, 15 million people suffer a stroke each year – that's one every nine minutes in Canada and every 90 seconds in the United States. The results can be devastating. Ischemic stroke, the most common, is caused by a clot in a blood vessel in the brain. The sudden loss of blood flow causes brain cells to die, which can permanently affect speech, vision, balance and movement.

The international trial enrolled 1,105 patients between March 2017 and August 2019 at centres in North America, Europe, Australia, and Asia – a global academic collaboration bringing together scientists, clinicians, funding agencies, and industry.

"The collaboration between NoNO Inc., the University of Calgary and investigators at 48 leading stroke hospitals around the world has shown how effective such an academic-industry partnership can be in running high-quality, foundational stroke trials that can lead to positive changes in clinical practice," says Dr. Michael Tymianski, MD, PhD, CEO of NoNO Inc. and the inventor of nerinetide.

The results in the current study, called the ESCAPE-NA1 Trial, build on the success of the ESCAPE trial, in which the Calgary Stroke Program proved that a clot retrieval procedure known as EVT can dramatically improve patient outcomes after an acute ischemic stroke. During the procedure, a catheter is inserted in the groin and guided through blood vessels into the brain. A tiny metal mesh device is used to grab the clot and pull it out. The current study investigates whether administering nerinetide in addition to clot retrieval improves the patient's ability to recover.

Source:

University of Calgary

Journal reference:

Hill, M.D, et al. (2020) Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke (ESCAPE-NA1): a multicentre, double-blind, randomised controlled trial. The Lancet. doi.org/10.1016/S0140-6736(20)30258-0.

Research from Rutgers Cancer Institute of New Jersey shows administering the immunotherapy drug pembrolizumab together with chemotherapy given at the same time as radiation treatment (chemoradiation) is safe and tolerable as a first-line therapy for patients with stage 3 non-small cell lung cancer (NSCLC). The work, stemming from a multi-center phase 1 clinical trial led by Rutgers Cancer Institute, is published in the February 20 online edition of JAMA Oncology.

“Locally advanced NSCLC accounts for 20 to 25 percent of all new diagnoses of NSCLC, with five-year overall survival rates of between 25 to 30 percent when standard therapy is given. Current standard treatment in which an immunotherapy drug is administered after chemoradiaton offers a 57 percent progression-free survival rate compared to 43.5 percent when chemoradiation is given alone. Our team wanted to examine the safety and tolerability of the immunotherapy drug pembrolizumab when administered concurrently with chemoradiation, as we’ve learned from first-line treatment of stage 4 disease that we see better patient outcomes the earlier immunotherapy is given,” shares Rutgers Cancer Institute radiation oncologist Salma Jabbour, MD, who is the lead and corresponding author of the current work.

Typically, the human body’s immune system recognizes abnormal cells in the body and destroys them. Cancer cells frequently create proteins (PD-L1, programmed cell death ligand-1) on the cell surface that act as signals to turn off this part of the immune system. Pembrolizumab is a drug approved by the Food and Drug Administration to treat melanoma and other forms of cancer that targets PD-1 receptors, which act as a signaling ‘switch.’ Pembrolizumab blocks this action and turns the ‘switch’ back on, allowing the immune system to recognize cancer cells as foreign and attack them.

For a 27 month period between 2016 and 2018, 23 participants were enrolled (52 percent were women; median age 69 years). Five cohorts evaluating different timing and dosing of pembrolizumab combined with chemotherapy (carboplatin and paclitaxel weekly) and definitive radiation therapy (60 Gy in 2 Gy/day x 30 fractions) for unresectable, locally advanced, stage 3 disease were examined. Median follow-up time was 16 months.

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Results show the combined treatment is feasible and well tolerated with a 12-month progression-free survival of 69.7 percent. Clinical benefit accounted for 94.6 percent at a median of 12.6 months. Of 19 evaluable patients (those who received 2 or more cycles of pembrolizumab) for response, the best response to therapy was a partial response seen in 73.7 percent, followed by 15.8 percent with a complete response, and 5.3 percent with stable disease. Local progression occurred in one patient, and of the six who developed metastatic disease, the median time to metastatic disease was 14.7 months. While there was an increased rate of pneumonitis, the authors note that patients with this form of lung inflammation responded to high-dose steroid treatment.

This study demonstrates that the combination of immunotherapy with chemoradiation has the potential to improve cure rates for patients with stage 3 non-small cell lung cancer.”

Dr. Salma Jabbour, professor of radiation oncology at Rutgers Robert Wood Johnson Medical School

Given the risk of pneumonitis when pembrolizumab is given with chemoradiation, the authors note further evaluation of the treatment combination through clinical trials is warranted, where careful radiation design to limit key lung parameters and biomarkers can be implemented. They add study limitations include the small sample size and limited follow-up duration.

Source:

Rutgers Cancer Institute of New Jersey

Journal reference:

Jabbour, S.K, et al. (2020) Phase 1 Trial of Pembrolizumab Administered Concurrently With Chemoradiotherapy for Locally Advanced Non–Small Cell Lung Cancer. JAMA Oncology. doi.org/10.1001/jamaoncol.2019.6731.